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Sample records for antibody dependent enhancement

  1. Enhanced antibody-dependent cellular phagocytosis by chimeric monoclonal antibodies with tandemly repeated Fc domains.

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    Nagashima, Hiroaki; Ootsubo, Michiko; Fukazawa, Mizuki; Motoi, Sotaro; Konakahara, Shu; Masuho, Yasuhiko

    2011-04-01

    We previously reported that chimeric monoclonal antibodies (mAbs) with tandemly repeated Fc domains, which were developed by introducing tandem repeats of Fc domains downstream of 2 Fab domains, augmented binding avidities for all Fcγ receptors, resulting in enhanced antibody (Ab)-dependent cellular cytotoxicity. Here we investigated regarding Ab-dependent cellular phagocytosis (ADCP) mediated by these chimeric mAbs, which is considered one of the most important mechanisms that kills tumor cells, using two-color flow cytometric methods. ADCP mediated by T3-Ab, a chimeric mAb with 3 tandemly repeated Fc domains, was 5 times more potent than that by native anti-CD20 M-Ab (M-Ab hereafter). Furthermore, T3-Ab-mediated ADCP was resistant to competitive inhibition by intravenous Ig (IVIG), although M-Ab-mediated ADCP decreased in the presence of IVIG. An Fcγ receptor-blocking study demonstrated that T3-Ab mediated ADCP via both FcγRIA and FcγRIIA, whereas M-Ab mediated ADCP exclusively via FcγRIA. These results suggest that chimeric mAbs with tandemly repeated Fc domains enhance ADCP as well as ADCC, and that Fc multimerization may significantly enhance the efficacy of therapeutic Abs. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  2. Antibody-Dependent Enhancement of Dengue Virus Growth in Human Monocytes as a Risk Factor for Dengue Hemorrhagic Fever

    Science.gov (United States)

    1989-01-01

    Clamfication) Antibody-Dependent Enhancement of Dengue Virus Growth in Human Monocytes as a Risk Factor for Dengue Hemorrhagic Fever 𔃼 PERSONAL AjTHOR(S...FELD GROUP SUBGROUP Antibody-Dependent Enhancement of Dengue Virus Growth in Human Monocytes as a Risk Factor for Dengue Hemorrhagic Fever . 19...clinically diagnosed as dengue hemorrhagic fever . Antibody-dependent enhancement of virus growth was quantitated by measurement of virus yields in

  3. Antibody-dependent enhancement of dengue virus infection is inhibited by SA-17, a doxorubicin derivative

    NARCIS (Netherlands)

    Ayala Nunez, Vanesa; Jarupathirun, Patsaporn; Kaptein, Suzanne; Neyts, Johan; Smit, Jolanda

    2013-01-01

    Antibody-dependent enhancement (ADE) is thought to play a critical role in the exacerbation of dengue virus (DENV)-induced disease during a heterologous re-infection. Despite ADE's clinical impact, only a few antiviral compounds have been assessed for their anti-ADE activity. We reported earlier

  4. Anti-Idiotypic Antibodies Specific to prM Monoantibody Prevent Antibody Dependent Enhancement of Dengue Virus Infection.

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    Wang, Miao; Yang, Fan; Huang, Dana; Huang, Yalan; Zhang, Xiaomin; Wang, Chao; Zhang, Shaohua; Zhang, Renli

    2017-01-01

    Dengue virus (DENV) co-circulates as four serotypes (DENV1-4). Primary infection only leads to self-limited dengue fever. But secondary infection with another serotype carries a higher risk of increased disease severity, causing life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Serotype cross-reactive antibodies facilitate DENV infection in Fc-receptor-bearing cells by promoting virus entry via Fcγ receptors (FcγR), a process known as antibody dependent enhancement (ADE). Most studies suggested that enhancing antibodies were mainly specific to the structural premembrane protein (prM) of DENV. However, there is still no effective drugs or vaccines to prevent ADE. In this study, we firstly confirmed that both DENV-2 infected human sera (anti-DENV-2) and DENV-2 prM monoclonal antibody (prM mAb) could significantly enhance DENV-1 infection in K562 cells. Then we developed anti-idiotypic antibodies (prM-AIDs) specific to prM mAb by immunizing of Balb/c mice. Results showed that these polyclonal antibodies can dramatically reduce ADE phenomenon of DENV-1 infection in K562 cells. To further confirm the anti-ADE effect of prM-AIDs in vivo , interferon-α and γ receptor-deficient mice (AG6) were used as the mouse model for DENV infection. We found that administration of DENV-2 prM mAb indeed caused a higher DENV-1 titer as well as interleukin-10 (IL-10) and alaninea minotransferase (ALT) in mice infected with DENV-1, similar to clinical ADE symptoms. But when we supplemented prM-AIDs to DENV-1 challenged AG6 mice, the viral titer, IL-10 and ALT were obviously decreased to the negative control level. Of note, the number of platelets in peripheral blood of prM-AIDs group were significantly increased at day 3 post infection with DENV-1 compared that of prM-mAb group. These results confirmed that our prM-AIDs could prevent ADE not only in vitro but also in vivo , suggested that anti-idiotypic antibodies might be a new choice to be considered to treat

  5. Anti-Idiotypic Antibodies Specific to prM Monoantibody Prevent Antibody Dependent Enhancement of Dengue Virus Infection

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    Miao Wang

    2017-05-01

    Full Text Available Dengue virus (DENV co-circulates as four serotypes (DENV1-4. Primary infection only leads to self-limited dengue fever. But secondary infection with another serotype carries a higher risk of increased disease severity, causing life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS. Serotype cross-reactive antibodies facilitate DENV infection in Fc-receptor-bearing cells by promoting virus entry via Fcγ receptors (FcγR, a process known as antibody dependent enhancement (ADE. Most studies suggested that enhancing antibodies were mainly specific to the structural premembrane protein (prM of DENV. However, there is still no effective drugs or vaccines to prevent ADE. In this study, we firstly confirmed that both DENV-2 infected human sera (anti-DENV-2 and DENV-2 prM monoclonal antibody (prM mAb could significantly enhance DENV-1 infection in K562 cells. Then we developed anti-idiotypic antibodies (prM-AIDs specific to prM mAb by immunizing of Balb/c mice. Results showed that these polyclonal antibodies can dramatically reduce ADE phenomenon of DENV-1 infection in K562 cells. To further confirm the anti-ADE effect of prM-AIDs in vivo, interferon-α and γ receptor-deficient mice (AG6 were used as the mouse model for DENV infection. We found that administration of DENV-2 prM mAb indeed caused a higher DENV-1 titer as well as interleukin-10 (IL-10 and alaninea minotransferase (ALT in mice infected with DENV-1, similar to clinical ADE symptoms. But when we supplemented prM-AIDs to DENV-1 challenged AG6 mice, the viral titer, IL-10 and ALT were obviously decreased to the negative control level. Of note, the number of platelets in peripheral blood of prM-AIDs group were significantly increased at day 3 post infection with DENV-1 compared that of prM-mAb group. These results confirmed that our prM-AIDs could prevent ADE not only in vitro but also in vivo, suggested that anti-idiotypic antibodies might be a new choice to be considered to

  6. Antibody-Dependent Enhancement of Dengue Virus Infection in Primary Human Macrophages; Balancing Higher Fusion against Antiviral Responses

    NARCIS (Netherlands)

    Flipse, Jacky; Diosa-Toro, Mayra A.; Hoornweg, Tabitha E.; van de Pol, Denise P. I.; Urcuqui-Inchima, Silvio; Smit, Jolanda M.

    2016-01-01

    The dogma is that the human immune system protects us against pathogens. Yet, several viruses, like dengue virus, antagonize the hosts' antibodies to enhance their viral load and disease severity; a phenomenon called antibody-dependent enhancement of infection. This study offers novel insights in

  7. Epidemics with Multistrain Interactions: Cross Immunity and Antibody-Dependent Enhancement

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    Bianco, Simone; Shaw, Leah

    2008-03-01

    Dynamics of epidemic spread is a problem of global interest. In this work we investigate the dynamical properties of a multistrain disease in a population where the strains interact via antibody-dependent enhancement (ADE) and cross immunity. ADE is a property of some multistrain diseases, such as dengue fever and Ebola, in which the antibodies generated by a primary infection with a strain tend to increase the infectiousness of a secondary infection with a different strain. After a primary infection, cross immunity provides temporary reduced susceptibility to the other strains. The presence of chaotic outbreaks and desynchronization between strains has already been observed in a model with no cross immunity if the ADE is sufficiently strong. The addition of weak cross immunity provides a stabilizing effect, while strong cross immunity leads to large amplitude chaotic outbreaks. A stochastic version of the model is also considered.

  8. Ocaratuzumab, an Fc-engineered antibody demonstrates enhanced antibody-dependent cell-mediated cytotoxicity in chronic lymphocytic leukemia.

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    Cheney, Carolyn M; Stephens, Deborah M; Mo, Xiaokui; Rafiq, Sarwish; Butchar, Jonathan; Flynn, Joseph M; Jones, Jeffrey A; Maddocks, Kami; O'Reilly, Adrienne; Ramachandran, Abhijit; Tridandapani, Susheela; Muthusamy, Natarajan; Byrd, John C

    2014-01-01

    Chronic lymphocytic leukemia (CLL) is common in both developed and developing nations where the need for inexpensive and convenient administration of therapy is apparent. Ocaratuzumab is a novel Fc-engineered humanized IgG1 anti-CD20 monoclonal antibody (mAb) designed for effective antibody-dependent cell-mediated cytotoxicity (ADCC) at very low concentrations that may facilitate sub-cutaneous (vs. intravenous) dosing. Here, we report ocaratuzumab's potency against CLL cells. In vitro assessment of ocaratuzumab's direct cytotoxicity (DC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and ADCC was performed on CLL cells. Ocaratuzumab induced DC, CDC, and ADCP similarly to rituximab or ofatumumab (anti-CD20 mAbs). However, ocaratuzumab showed an advantage in NK cell-mediated ADCC over these antibodies. In allogeneic ADCC, [E:T (effector:target) ratios = 25:1, 12:1, 6:1], ocaratuzumab (10 µg/mL) improved ADCC by ~3-fold compared with rituximab or ofatumumab (P<0.001 all tested E:T ratios). Notably, the superiority of ocaratuzumab-induced ADCC was observed at low concentrations (0.1-10 ug/ml; P<0.03; allogeneic assays). In extended allogeneic ADCC E:T titration, ocaratuzumab (0.1 µg/mL) demonstrated 19.4% more cytotoxicity than rituximab (E:T = 0.38:1; P = 0.0066) and 21.5% more cytotoxicity than ofatumumab (E:T = 1.5:1; P = 0.0015). In autologous ADCC, ocaratuzumab (10 µg/mL) demonstrated ~1.5-fold increase in cytotoxicity compared with rituximab or ofatumumab at all E:T ratios tested (E:Ts = 25:1,12:1,6:1; all P<0.001). Obinutuzumab, a glyco-engineered anti-CD20 mAb, showed no improvement in ADCC activity compared with ocaratuzumab. The enhanced ADCC of ocaratuzumab suggests that it may be effective at low concentrations. If supported by clinical investigation, this feature could potentially allow for subcutaneous dosing at low doses that could expand the potential of administering chemoimmunotherapy in developing

  9. Low Levels of Antibody-Dependent Enhancement in Vitro Using Viruses and Plasma from Dengue Patients

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    Chaichana, Panjaporn; Okabayashi, Tamaki; Puiprom, Orapim; Sasayama, Mikiko; Sasaki, Tadahiro; Yamashita, Akifumi; Ramasoota, Pongrama; Kurosu, Takeshi; Ikuta, Kazuyoshi

    2014-01-01

    Background The majority of dengue patients infected with any serotype of dengue virus (DENV) are asymptomatic, but the remainder may develop a wide spectrum of clinical symptoms, ranging from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF). Severe cases occur more often in patients who experience a secondary infection with a different virus serotype. A phenomenon called antibody-dependent enhancement (ADE) has been proposed to explain the onset of these severe cases, but the exact mechanism of ADE remains unclear. Methodology/Principal Finding Virus neutralization and ADE assays were performed using ultracentrifugation supernatants of acute-phase sera from patients with secondary infections or human monoclonal antibodies (HuMAbs) as anti-DENV antibodies. Virus sources included infectious serum-derived viruses from the ultracentrifugation precipitates, laboratory-culture adapted DENV, or recombinant DENVs derived from patient sera. In contrast to the high levels of ADE observed with laboratory virus strains, low ADE was observed with autologous patient-derived viruses, when patient sera were used to provide the antibody component in the ADE assays. Similar results were obtained using samples from DF and DHF patients. Recombinant-viruses derived from DHF patients showed only minor differences in neutralization and ADE activity in the presence of HuMAbs or plasma derived from the same DHF patient. Conclusion/Significance Serum or plasma taken from patients during the acute phase of a secondary infection showed high levels of ADE, but no neutralization activity, when assayed in the presence of laboratory-adapted virus strains. By contrast, serum or plasma from the same patient showed high levels of neutralization activity but failed to induce significant ADE when the assays were performed with autologous virus. These results demonstrate the significance of the virus source when measuring ADE. They also suggest that repeated passage of DENV in cell

  10. Enhancement of antibody-dependent cell mediated cytotoxicity: a new era in cancer treatment

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    Rajasekaran N

    2015-05-01

    Full Text Available Narendiran Rajasekaran,1,* Cariad Chester,1,* Atsushi Yonezawa,1,2 Xing Zhao,1,3 Holbrook E Kohrt1 1Division of Oncology, Stanford School of Medicine, Stanford University, Stanford, CA, USA; 2Department of Clinical Pharmacology and Therapeutics, Kyoto University Hospital, Kyoto, Japan; 3Tissue Engineering and Stem Cells Research Center, Department of Immunology, Guiyang Medical University, Guiyang, Guizhou Province, People's Republic of China *These authors contributed equally to this work Abstract: The therapeutic efficacy of some anti-tumor monoclonal antibodies (mAbs depends on the capacity of the mAb to recognize the tumor-associated antigen and induce cytotoxicity via a network of immune effector cells. This process of antibody-dependent cell-mediated cytotoxicity (ADCC against tumor cells is triggered by the interaction of the fragment crystallizable (Fc portion of the mAb with the Fc receptors on effector cells like natural killer cells, macrophages, γδ T cells, and dendritic cells. By augmenting ADCC, the antitumor activity of mAbs can be significantly increased. Currently, identifying and developing therapeutic agents that enhance ADCC is a growing area of research. Combining existing tumor-targeting mAbs and ADCC-promoting agents that stimulate effector cells will translate to greater clinical responses. In this review, we discuss strategies for enhancing ADCC and emphasize the potential of combination treatments that include US Food and Drug Administration-approved mAbs and immunostimulatory therapeutics. Keywords: ADCC, NK cell, reovirus, TLR, CD137

  11. Can we find a possible structural explanation for antibody-dependent enhancement of dengue virus infection resulting in hemorrhagic fever?

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    Mikita, Cecilia P; Padlan, Eduardo A

    2016-03-01

    Dengue virus infection is one of the most prevalent mosquito-borne illnesses worldwide, affecting as many as 400 million persons annually. Most people experience a self-limited viral illness, but some experience life-threatening disease. Subsequent infection with other dengue virus serotypes increases the risk of development of severe dengue disease with plasma leakage with or without hemorrhage and end organ impairment. Antibody-dependent enhancement of dengue virus infection has been implicated in the development of severe dengue disease, previously referred to as dengue hemorrhagic fever and dengue shock syndrome. We propose a structural explanation for the role of non-neutralizing antibodies in the development of antibody-dependent enhancement of dengue virus infection via complement fixation or binding to Fcγ receptors facilitating entry into target cells. Published by Elsevier Ltd.

  12. Manipulation of immunodominant dengue virus E protein epitopes reduces potential antibody-dependent enhancement

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    Hughes Holly R

    2012-06-01

    Full Text Available Abstract Background Dengue viruses (DENV are the most important arboviruses of humans and cause significant disease. Infection with DENV elicits antibody responses to the envelope glycoprotein, predominantly against immunodominant, cross-reactive, weakly-neutralizing epitopes. These weakly-neutralizing antibodies are implicated in enhancing infection via Fcγ receptor bearing cells and can lead to increased viral loads that are associated with severe disease. Here we describe results from the development and testing of cross-reactivity reduced DENV-2 DNA vaccine candidates that contain substitutions in immunodominant B cell epitopes of the fusion peptide and domain III of the envelope protein. Results Cross-reactivity reduced and wild-type vaccine candidates were similarly immunogenic in outbred mice and elicited high levels of neutralizing antibody, however mice immunized with cross-reactivity reduced vaccines produced significantly reduced levels of immunodominant cross-reactive antibodies. Sera from mice immunized with wild-type, fusion peptide-, or domain III- substitution containing vaccines enhanced heterologous DENV infection in vitro, unlike sera from mice immunized with a vaccine containing a combination of both fusion peptide and domain III substitutions. Passive transfer of immune sera from mice immunized with fusion peptide and domain III substitutions also reduced the development of severe DENV disease in AG129 mice when compared to mice receiving wild type immune sera. Conclusions Reducing cross-reactivity in the envelope glycoprotein of DENV may be an approach to improve the quality of the anti-DENV immune response.

  13. Substitution of the precursor peptide prevents anti-prM antibody-mediated antibody-dependent enhancement of dengue virus infection.

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    Wang, Ying; Si, Lu-Lu; Guo, Xiao-Lan; Cui, Guo-Hui; Fang, Dan-Yun; Zhou, Jun-Mei; Yan, Hui-Jun; Jiang, Li-Fang

    2017-02-02

    Antibody-dependent enhancement (ADE) is currently considered as the mechanism underlying the pathogenesis of severe dengue disease. Many studies have shown that precursor (pr) peptide-specific antibodies do not efficiently neutralize infection but potently promote ADE of dengue virus (DENV) infection. To explore the effect of pr peptide substitution on neutralization and ADE of DENV infection, the rabbit anti-prM polyclonal antibodies (pAbs) and anti-JEVpr/DENV-M pAbs were prepared, and the neutralization and ADE of these two pAbs were further compared. Here, we report that both anti-JEVpr/DENV-M and anti-prM pAbs exhibited broad cross-reactivity and only partial neutralization with four DENV serotypes and immature DENV. Rabbit anti-prM pAbs showed a significant enhancement in a broad range of serum dilutions. However, there was no statistically significant difference in the enhancing activity of rabbit anti-JEVpr/DENV-M pAbs at various levels of dilution. These results demonstrate that anti-prM antibody-mediated ADE can be prevented by JEV pr peptide replacement. The present study contribute further to research on the pathogenesis of DENV infection. Copyright © 2016. Published by Elsevier B.V.

  14. Modulation of Dengue/Zika Virus Pathogenicity by Antibody-Dependent Enhancement and Strategies to Protect Against Enhancement in Zika Virus Infection

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    Rekha Khandia

    2018-04-01

    Full Text Available Antibody-dependent enhancement (ADE is a phenomenon in which preexisting poorly neutralizing antibodies leads to enhanced infection. It is a serious concern with mosquito-borne flaviviruses such as Dengue virus (DENV and Zika virus (ZIKV. In vitro experimental evidences have indicated the preventive, as well as a pathogenicity-enhancing role, of preexisting DENV antibodies in ZIKV infections. ADE has been confirmed in DENV but not ZIKV infections. Principally, the Fc region of the anti-DENV antibody binds with the fragment crystallizable gamma receptor (FcγR, and subsequent C1q interactions and immune effector functions are responsible for the ADE. In contrast to normal DENV infections, with ADE in DENV infections, inhibition of STAT1 phosphorylation and a reduction in IRF-1 gene expression, NOS2 levels, and RIG-1 and MDA-5 expression levels occurs. FcγRIIA is the most permissive FcγR for DENV-ADE, and under hypoxic conditions, hypoxia-inducible factor-1 alpha transcriptionally enhances expression levels of FcγRIIA, which further enhances ADE. To produce therapeutic antibodies with broad reactivity to different DENV serotypes, as well as to ZIKV, bispecific antibodies, Fc region mutants, modified Fc regions, and anti-idiotypic antibodies may be engineered. An in-depth understanding of the immunological and molecular mechanisms of DENV-ADE of ZIKV pathogenicity will be useful for the design of common and safe therapeutics and prophylactics against both viral pathogens. The present review discusses the role of DENV antibodies in modulating DENV/ZIKV pathogenicity/infection and strategies to counter ADE to protect against Zika infection.

  15. Pichia pastoris-Expressed Bivalent Virus-Like Particulate Vaccine Induces Domain III-Focused Bivalent Neutralizing Antibodies without Antibody-Dependent Enhancement in Vivo

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    Rahul Shukla

    2018-01-01

    Full Text Available Dengue, a significant public health problem in several countries around the world, is caused by four different serotypes of mosquito-borne dengue viruses (DENV-1, -2, -3, and -4. Antibodies to any one DENV serotype which can protect against homotypic re-infection, do not offer heterotypic cross-protection. In fact, cross-reactive antibodies may augment heterotypic DENV infection through antibody-dependent enhancement (ADE. A recently launched live attenuated vaccine (LAV for dengue, which consists of a mixture of four chimeric yellow-fever/dengue vaccine viruses, may be linked to the induction of disease-enhancing antibodies. This is likely related to viral interference among the replicating viral strains, resulting in an unbalanced immune response, as well as to the fact that the LAV encodes prM, a DENV protein documented to elicit ADE-mediating antibodies. This makes it imperative to explore the feasibility of alternate ADE risk-free vaccine candidates. Our quest for a non-replicating vaccine centered on the DENV envelope (E protein which mediates virus entry into the host cell and serves as an important target of the immune response. Serotype-specific neutralizing epitopes and the host receptor recognition function map to E domain III (EDIII. Recently, we found that Pichia pastoris-expressed DENV E protein, of all four serotypes, self-assembled into virus-like particles (VLPs in the absence of prM. Significantly, these VLPs displayed EDIII and elicited EDIII-focused DENV-neutralizing antibodies in mice. We now report the creation and characterization of a novel non-replicating recombinant particulate vaccine candidate, produced by co-expressing the E proteins of DENV-1 and DENV-2 in P. pastoris. The two E proteins co-assembled into bivalent mosaic VLPs (mVLPs designated as mE1E2bv VLPs. The mVLP, which preserved the serotype-specific antigenic integrity of its two component proteins, elicited predominantly EDIII-focused homotypic virus

  16. Immunomodulating actions of carotenoids: enhancement of in vivo and in vitro antibody production to T-dependent antigens.

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    Jyonouchi, H; Zhang, L; Gross, M; Tomita, Y

    1994-01-01

    Previously, we demonstrated an enhancement of in vitro antibody (Ab) production in response to T-dependent antigens (TD-Ag) by astaxanthin, a carotenoid without vitamin A activity. The effects of beta-carotene, a carotenoid with vitamin A activity, and lutein, another carotenoid without vitamin A activity, on in vitro Ab production were examined with spleen cells from young and old B6 mice. In addition, the in vivo effects of lutein, astaxanthin, and beta-carotene on Ab production were studied in young and old B6 mice. Lutein, but not beta-carotene, enhanced in vitro Ab production in response to TD-Ags. The depletion of T-helper cells prevented the enhancement of Ab production by lutein and astaxanthin. In vivo Ab production in response to TD-Ag was significantly enhanced by lutein, astaxanthin, and beta-carotene. The numbers of immunoglobulin M- and G-secreting cells also increased in vivo with the administration of these carotenoids when mice were primed with TD-Ags. Antibody production in response to TD-Ags in vivo and in vitro was significantly lower in old than in young B6 mice. Astaxanthin supplements partially restored decreased in vivo Ab production in response to TD-Ags in old B6 mice. Lutein and beta-carotene also enhanced in vivo Ab production in response to TD-Ags in old B6 mice, although to a lesser extent than did astaxanthin. However, none of the carotenoids had an effect on in vivo or in vitro Ab production in response to T-independent antigen. These results indicate significant immunomodulating actions of carotenoids for humoral immune responses to TD-Ags and suggest that carotenoid supplementation may be beneficial in restoring humoral immune responses in older animals.

  17. Lipidated dengue-2 envelope protein domain III independently stimulates long-lasting neutralizing antibodies and reduces the risk of antibody-dependent enhancement.

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    Chen-Yi Chiang

    Full Text Available BACKGROUND: Dengue virus is a mosquito-transmitted virus that can cause self-limiting dengue fever, severe life-threatening dengue hemorrhagic fever and dengue shock syndrome. The existence of four serotypes of dengue virus has complicated the development of an effective and safe dengue vaccine. Recently, a clinical phase 2b trial of Sanofi Pasteur's CYD tetravalent dengue vaccine revealed that the vaccine did not confer full protection against dengue-2 virus. New approaches to dengue vaccine development are urgently needed. Our approach represents a promising method of dengue vaccine development and may even complement the deficiencies of the CYD tetravalent dengue vaccine. METHODOLOGY/PRINCIPAL FINDINGS: Two important components of a vaccine, the immunogen and immunopotentiator, were combined into a single construct to generate a new generation of vaccines. We selected dengue-2 envelope protein domain III (D2ED III as the immunogen and expressed this protein in lipidated form in Escherichia coli, yielding an immunogen with intrinsic immunopotentiation activity. The formulation containing lipidated D2ED III (LD2ED III in the absence of exogenous adjuvant elicited higher D2ED III-specific antibody responses than those obtained from its nonlipidated counterpart, D2ED III, and dengue-2 virus. In addition, the avidity and neutralizing capacity of the antibodies induced by LD2ED III were higher than those elicited by D2ED III and dengue-2 virus. Importantly, we showed that after lipidation, the subunit candidate LD2ED III exhibited increased immunogenicity while reducing the potential risk of antibody-dependent enhancement of infection in mice. CONCLUSIONS/SIGNIFICANCE: Our study suggests that the lipidated subunit vaccine approach could be applied to other serotypes of dengue virus and other pathogens.

  18. Mosquito Saliva Increases Endothelial Permeability in the Skin, Immune Cell Migration, and Dengue Pathogenesis during Antibody-Dependent Enhancement.

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    Michael A Schmid

    2016-06-01

    Full Text Available Dengue remains the most prevalent arthropod-borne viral disease in humans. While probing for blood vessels, Aedes aegypti and Ae. albopictus mosquitoes transmit the four serotypes of dengue virus (DENV1-4 by injecting virus-containing saliva into the skin. Even though arthropod saliva is known to facilitate transmission and modulate host responses to other pathogens, the full impact of mosquito saliva on dengue pathogenesis is still not well understood. Inoculating mice lacking the interferon-α/β receptor intradermally with DENV revealed that mosquito salivary gland extract (SGE exacerbates dengue pathogenesis specifically in the presence of enhancing serotype-cross-reactive antibodies-when individuals already carry an increased risk for severe disease. We further establish that SGE increases viral titers in the skin, boosts antibody-enhanced DENV infection of dendritic cells and macrophages in the dermis, and amplifies dendritic cell migration to skin-draining lymph nodes. We demonstrate that SGE directly disrupts endothelial barrier function in vitro and induces endothelial permeability in vivo in the skin. Finally, we show that surgically removing the site of DENV transmission in the skin after 4 hours rescued mice from disease in the absence of SGE, but no longer prevented lethal antibody-enhanced disease when SGE was present. These results indicate that SGE accelerates the dynamics of dengue pathogenesis after virus transmission in the skin and induces severe antibody-enhanced disease systemically. Our study reveals novel aspects of dengue pathogenesis and suggests that animal models of dengue and pre-clinical testing of dengue vaccines should consider mosquito-derived factors as well as enhancing antibodies.

  19. Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines

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    Cavazzoni Andrea

    2012-12-01

    Full Text Available Abstract Background The epidermal growth factor receptor (EGFR is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. Results In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. Conclusion Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib.

  20. Fcγ-receptor IIa-mediated Src Signaling Pathway Is Essential for the Antibody-Dependent Enhancement of Ebola Virus Infection.

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    Wakako Furuyama

    2016-12-01

    Full Text Available Antibody-dependent enhancement (ADE of Ebola virus (EBOV infection has been demonstrated in vitro, raising concerns about the detrimental potential of some anti-EBOV antibodies. ADE has been described for many viruses and mostly depends on the cross-linking of virus-antibody complexes to cell surface Fc receptors, leading to enhanced infection. However, little is known about the molecular mechanisms underlying this phenomenon. Here we show that Fcγ-receptor IIa (FcγRIIa-mediated intracellular signaling through Src family protein tyrosine kinases (PTKs is required for ADE of EBOV infection. We found that deletion of the FcγRIIa cytoplasmic tail abolished EBOV ADE due to decreased virus uptake into cellular endosomes. Furthermore, EBOV ADE, but not non-ADE infection, was significantly reduced by inhibition of the Src family protein PTK pathway, which was also found to be important to promote phagocytosis/macropinocytosis for viral uptake into endosomes. We further confirmed a significant increase of the Src phosphorylation mediated by ADE. These data suggest that antibody-EBOV complexes bound to the cell surface FcγRIIa activate the Src signaling pathway that leads to enhanced viral entry into cells, providing a novel perspective for the general understanding of ADE of virus infection.

  1. Enhancement of antibody-dependent cell-mediated cytotoxicity by endowing IgG with FcαRI (CD89) binding.

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    Borrok, M Jack; Luheshi, Nadia M; Beyaz, Nurten; Davies, Gareth C; Legg, James W; Wu, Herren; Dall'Acqua, William F; Tsui, Ping

    2015-01-01

    Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are crucial to the efficacy of many antibody therapeutics. In addition to IgG, antibodies of the IgA isotype can also promote cell killing through engagement of myeloid lineage cells via interactions between the IgA-Fc and FcαRI (CD89). Herein, we describe a unique, tandem IgG1/IgA2 antibody format in the context of a trastuzumab variable domain that exhibits enhanced ADCC and ADCP capabilities. The IgG1/IgA2 tandem Fc format retains IgG1 FcγR binding as well as FcRn-mediated serum persistence, yet is augmented with myeloid cell-mediated effector functions via FcαRI/IgA Fc interactions. In this work, we demonstrate anti-human epidermal growth factor receptor-2 antibodies with the unique tandem IgG1/IgA2 Fc can better recruit and engage cytotoxic polymorphonuclear (PMN) cells than either the parental IgG1 or IgA2. Pharmacokinetics of IgG1/IgA2 in BALB/c mice are similar to the parental IgG, and far surpass the poor serum persistence of IgA2. The IgG1/IgA2 format is expressed at similar levels and with similar thermal stability to IgG1, and can be purified via standard protein A chromatography. The tandem IgG1/IgA2 format could potentially augment IgG-based immunotherapeutics with enhanced PMN-mediated cytotoxicity while avoiding many of the problems associated with developing IgAs.

  2. Dengue virus specific IgY provides protection following lethal dengue virus challenge and is neutralizing in the absence of inducing antibody dependent enhancement.

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    Ashley L Fink

    2017-07-01

    Full Text Available Dengue hemorrhagic fever (DHF and dengue shock syndrome (DSS are severe disease manifestations that can occur following sequential infection with different dengue virus serotypes (DENV1-4. At present, there are no licensed therapies to treat DENV-induced disease. DHF and DSS are thought to be mediated by serotype cross-reactive antibodies that facilitate antibody-dependent enhancement (ADE by binding to viral antigens and then Fcγ receptors (FcγR on target myeloid cells. Using genetically engineered DENV-specific antibodies, it has been shown that the interaction between the Fc portion of serotype cross-reactive antibodies and FcγR is required to induce ADE. Additionally, it was demonstrated that these antibodies were as neutralizing as their non-modified variants, were incapable of inducing ADE, and were therapeutic following a lethal, antibody-enhanced infection. Therefore, we hypothesized that avian IgY, which do not interact with mammalian FcγR, would provide a novel therapy for DENV-induced disease. We demonstrate here that goose-derived anti-DENV2 IgY neutralized DENV2 and did not induce ADE in vitro. Anti-DENV2 IgY was also protective in vivo when administered 24 hours following a lethal DENV2 infection. We were also able to demonstrate via epitope mapping that both full-length and alternatively spliced anti-DENV2 IgY recognized different epitopes, including epitopes that have not been previously identified. These observations provide evidence for the potential therapeutic applications of goose-derived anti-DENV2 IgY.

  3. Synthetic multivalent V3 glycopeptides display enhanced recognition by glycan-dependent HIV-1 broadly neutralizing antibodies.

    Science.gov (United States)

    Cai, Hui; Orwenyo, Jared; Guenaga, Javier; Giddens, John; Toonstra, Christian; Wyatt, Richard T; Wang, Lai-Xi

    2017-05-14

    We describe here the synthesis of novel multivalent HIV V3 domain glycopeptides and their binding to broadly neutralizing antibodies PGT128 and 10-1074. Our binding data reveal a distinct mode of antigen recognition by the two antibodies and further suggest that multivalent glycopeptides could mimic the neutralizing epitopes more efficiently than the monomeric glycopeptide.

  4. Immunological low-dose radiation modulates the pediatric medulloblastoma antigens and enhances antibody-dependent cellular cytotoxicity.

    Science.gov (United States)

    Das, Arabinda; McDonald, Daniel; Lowe, Stephen; Bredlau, Amy-Lee; Vanek, Kenneth; Patel, Sunil J; Cheshier, Samuel; Eskandari, Ramin

    2017-03-01

    Immunotherapy can be an effective treatment for pediatric medulloblastoma (MB) patients. However, major subpopulations do not respond to immunotherapy, due to the lack of antigenic mutations or the immune-evasive properties of MB cells. Clinical observations suggest that radiation therapy (RT) may expand the therapeutic reach of immunotherapy. The aim of the present investigation is to study the effect of low-dose X-ray radiation (LDXR, 1 Gy) on the functional immunological responses of MB cells (DAOY, D283, and D341). Induction of MB cell death was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Production of reactive oxygen species (ROS) was measured by fluorescent probes. Changes in the expression of  human leukocyte antigen (HLA) molecules and caspase-3 activities during treatment were analyzed using Western blotting and caspase-3 assay. Western blot analysis demonstrated that LDXR upregulated the expression of HLA class I and HLA II molecules by more than 20% compared with control and high-dose (12 Gy) groups in vitro. Several of these HLA subtypes, such as MAGE C1, CD137, and ICAM-1, have demonstrated upregulation. In addition, LDXR increases ROS production in association with phosphorylation of NF-κB and cell surface expression of mAb target molecules (HER2 and VEGF). These data suggest that a combined LDXR and mAb therapy can create a synergistic effect in vitro. These results suggest that LDXR modulates HLA molecules, leading to alterations in T-cell/tumor-cell interaction and enhancement of T-cell-mediated MB cell death. Also, low-dose radiotherapy combined with monoclonal antibody therapy may one day augment the standard treatment for MB, but more investigation is needed to prove its utility as a new therapeutic combination for MB patients.

  5. MEDI-563, a humanized anti-IL-5 receptor alpha mAb with enhanced antibody-dependent cell-mediated cytotoxicity function.

    Science.gov (United States)

    Kolbeck, Roland; Kozhich, Alexander; Koike, Masamichi; Peng, Li; Andersson, Cecilia K; Damschroder, Melissa M; Reed, Jennifer L; Woods, Robert; Dall'acqua, William W; Stephens, Geoffrey L; Erjefalt, Jonas S; Bjermer, Leif; Humbles, Alison A; Gossage, David; Wu, Herren; Kiener, Peter A; Spitalny, George L; Mackay, Charles R; Molfino, Nestor A; Coyle, Anthony J

    2010-06-01

    Peripheral blood eosinophilia and lung mucosal eosinophil infiltration are hallmarks of bronchial asthma. IL-5 is a critical cytokine for eosinophil maturation, survival, and mobilization. Attempts to target eosinophils for the treatment of asthma by means of IL-5 neutralization have only resulted in partial removal of airway eosinophils, and this warrants the development of more effective interventions to further explore the role of eosinophils in the clinical expression of asthma. We sought to develop a novel humanized anti-IL-5 receptor alpha (IL-5Ralpha) mAb with enhanced effector function (MEDI-563) that potently depletes circulating and tissue-resident eosinophils and basophils for the treatment of asthma. We used surface plasmon resonance to determine the binding affinity of MEDI-563 to FcgammaRIIIa. Primary human eosinophils and basophils were used to demonstrate antibody-dependent cell-mediated cytotoxicity. The binding epitope of MEDI-563 on IL-5Ralpha was determined by using site-directed mutagenesis. The consequences of MEDI-563 administration on peripheral blood and bone marrow eosinophil depletion was investigated in nonhuman primates. MEDI-563 binds to an epitope on IL-5Ralpha that is in close proximity to the IL-5 binding site, and it inhibits IL-5-mediated cell proliferation. MEDI-563 potently induces antibody-dependent cell-mediated cytotoxicity of both eosinophils (half-maximal effective concentration = 0.9 pmol/L) and basophils (half-maximal effective concentration = 0.5 pmol/L) in vitro. In nonhuman primates MEDI-563 depletes blood eosinophils and eosinophil precursors in the bone marrow. MEDI-563 might provide a novel approach for the treatment of asthma through active antibody-dependent cell-mediated depletion of eosinophils and basophils rather than through passive removal of IL-5. Copyright (c) 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  6. Secondary infection as a risk factor for dengue hemorrhagic fever/dengue shock syndrome: an historical perspective and role of antibody-dependent enhancement of infection.

    Science.gov (United States)

    Guzman, Maria G; Alvarez, Mayling; Halstead, Scott B

    2013-07-01

    Today, dengue viruses are the most prevalent arthropod-borne viruses in the world. Since the 1960s, numerous reports have identified a second heterologous dengue virus (DENV) infection as a principal risk factor for severe dengue disease (dengue hemorrhagic fever/dengue shock syndrome, DHF/DSS). Modifiers of dengue disease response include the specific sequence of two DENV infections, the interval between infections, and contributions from the human host, such as age, ethnicity, chronic illnesses and genetic background. Antibody-dependent enhancement (ADE) of dengue virus infection has been proposed as the early mechanism underlying DHF/DSS. Dengue cross-reactive antibodies raised following a first dengue infection combine with a second infecting virus to form infectious immune complexes that enter Fc-receptor-bearing cells. This results in an increased number of infected cells and increased viral output per cell. At the late illness stage, high levels of cytokines, possibly the result of T cell elimination of infected cells, result in vascular permeability, leading to shock and death. This review is focused on the etiological role of secondary infections (SI) and mechanisms of ADE.

  7. Dengue virus antibodies enhance Zika virus infection.

    Science.gov (United States)

    Paul, Lauren M; Carlin, Eric R; Jenkins, Meagan M; Tan, Amanda L; Barcellona, Carolyn M; Nicholson, Cindo O; Michael, Scott F; Isern, Sharon

    2016-12-01

    For decades, human infections with Zika virus (ZIKV), a mosquito-transmitted flavivirus, were sporadic, associated with mild disease, and went underreported since symptoms were similar to other acute febrile diseases. Recent reports of severe disease associated with ZIKV have greatly heightened awareness. It is anticipated that ZIKV will continue to spread in the Americas and globally where competent Aedes mosquito vectors are found. Dengue virus (DENV), the most common mosquito-transmitted human flavivirus, is both well-established and the source of outbreaks in areas of recent ZIKV introduction. DENV and ZIKV are closely related, resulting in substantial antigenic overlap. Through antibody-dependent enhancement (ADE), anti-DENV antibodies can enhance the infectivity of DENV for certain classes of immune cells, causing increased viral production that correlates with severe disease outcomes. Similarly, ZIKV has been shown to undergo ADE in response to antibodies generated by other flaviviruses. We tested the neutralizing and enhancing potential of well-characterized broadly neutralizing human anti-DENV monoclonal antibodies (HMAbs) and human DENV immune sera against ZIKV using neutralization and ADE assays. We show that anti-DENV HMAbs, cross-react, do not neutralize, and greatly enhance ZIKV infection in vitro . DENV immune sera had varying degrees of neutralization against ZIKV and similarly enhanced ZIKV infection. Our results suggest that pre-existing DENV immunity may enhance ZIKV infection in vivo and may lead to increased disease severity. Understanding the interplay between ZIKV and DENV will be critical in informing public health responses and will be particularly valuable for ZIKV and DENV vaccine design and implementation strategies.

  8. Molecular determinants of dengue virus 2 envelope protein important for virus entry in FcγRIIA-mediated antibody-dependent enhancement of infection

    Energy Technology Data Exchange (ETDEWEB)

    Chotiwan, Nunya; Roehrig, John T. [Arboviral Diseases Branch, Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Schlesinger, Jacob J. [Department of Medicine, University of Rochester, Rochester, NY 14642 (United States); Blair, Carol D. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Huang, Claire Y.-H., E-mail: yxh0@cdc.gov [Arboviral Diseases Branch, Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States)

    2014-05-15

    Antibody-dependent enhancement (ADE) of infection may cause severe illness in patients suffering a secondary infection by a heterologous dengue virus (DENV) serotype. During ADE of infection, cross-reactive non- or poorly-neutralizing antibodies form infectious virus-Ab complexes with the newly infecting serotype and enhance virus infection by binding to the Fcγ receptors (FcγR) on FcγR-bearing cells. In this study, we determined that molecular determinants of DENV2 envelope protein critical for virus entry during non-ADE infection are also required for ADE infection mediated by FcγRIIA, and binding of virus-Ab complexes with FcγRIIA alone is not sufficient for ADE of infection. The FcγRIIA mainly plays an auxiliary role in concentrating the virus–Ab complex to the cell surface, and other primary cellular receptors are required for virus entry. Understanding the viral entry pathway in ADE of DENV infection will greatly facilitate rational designs of anti-viral therapeutics against severe dengue disease associated with ADE. - Highlights: • KKK305/307/310 in DENV2 E-DIII is critical for virus attachment in ADE and non-ADE infection. • Binding of DENV2–Ab complex with FcγRII alone is not sufficient for virus entry in ADE infection. • Other primary receptors were required for DENV2 internalization during FcγRII–mediated ADE. • G104 and L135 of DENV2 E are critical for virus-mediated membrane fusion. • DENV2 virus-mediated membrane fusion is required for both ADE and non-ADE infection.

  9. Molecular determinants of dengue virus 2 envelope protein important for virus entry in FcγRIIA-mediated antibody-dependent enhancement of infection

    International Nuclear Information System (INIS)

    Chotiwan, Nunya; Roehrig, John T.; Schlesinger, Jacob J.; Blair, Carol D.; Huang, Claire Y.-H.

    2014-01-01

    Antibody-dependent enhancement (ADE) of infection may cause severe illness in patients suffering a secondary infection by a heterologous dengue virus (DENV) serotype. During ADE of infection, cross-reactive non- or poorly-neutralizing antibodies form infectious virus-Ab complexes with the newly infecting serotype and enhance virus infection by binding to the Fcγ receptors (FcγR) on FcγR-bearing cells. In this study, we determined that molecular determinants of DENV2 envelope protein critical for virus entry during non-ADE infection are also required for ADE infection mediated by FcγRIIA, and binding of virus-Ab complexes with FcγRIIA alone is not sufficient for ADE of infection. The FcγRIIA mainly plays an auxiliary role in concentrating the virus–Ab complex to the cell surface, and other primary cellular receptors are required for virus entry. Understanding the viral entry pathway in ADE of DENV infection will greatly facilitate rational designs of anti-viral therapeutics against severe dengue disease associated with ADE. - Highlights: • KKK305/307/310 in DENV2 E-DIII is critical for virus attachment in ADE and non-ADE infection. • Binding of DENV2–Ab complex with FcγRII alone is not sufficient for virus entry in ADE infection. • Other primary receptors were required for DENV2 internalization during FcγRII–mediated ADE. • G104 and L135 of DENV2 E are critical for virus-mediated membrane fusion. • DENV2 virus-mediated membrane fusion is required for both ADE and non-ADE infection

  10. Summary Report: Workshop on the Potential Risks of Antibody-Dependent Enhancement in Human HIV Vaccine Trials

    Science.gov (United States)

    1993-01-01

    disease course. gous flavivirus species) despite the fact that in vivo disease Lentiviraldiseases. Equine infectious anemia virus (EIAV) enhancement...Alan Schultz (Division of AIDS, NIAID), Herbert Smith (Food disease caused by equine infectious anemia virus. J Virol & Drug Administration), Gregory...of AIDS, National Institute of Allergy and Infectious Diseases. National Institutes of Health. Bethesda. Maryland. ’Health Sciences Division. the

  11. Subversion of early innate antiviral responses during antibody-dependent enhancement of Dengue virus infection induces severe disease in immunocompetent mice.

    Science.gov (United States)

    Costa, Vivian V; Fagundes, Caio T; Valadão, Deborah F; Ávila, Thiago V; Cisalpino, Daniel; Rocha, Rebeca F; Ribeiro, Lucas S; Ascenção, Fernando R; Kangussu, Lucas M; Celso, M Q; Astigarraga, Ruiz G; Gouveia, Frederico L; Silva, Tarcília A; Bonaventura, Daniela; Sampaio, Divaldo de Almeida; Leite, Ana Cristina L; Teixeira, Mauro M; Souza, Danielle G

    2014-08-01

    Dengue is a mosquito-borne disease caused by one of four serotypes of Dengue virus (DENV-1-4). Epidemiologic and observational studies demonstrate that the majority of severe dengue cases, dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS), occurs predominantly in either individuals with cross-reactive immunity following a secondary heterologous infection or in infants with primary DENV infections born from dengue-immune mothers, suggesting that B-cell-mediated and antibody responses impact on disease evolution. We demonstrate here that B cells play a pivotal role in host responses against primary DENV infection in mice. After infection, μMT(-/-) mice showed increased viral loads followed by severe disease manifestation characterized by intense thrombocytopenia, hemoconcentration, cytokine production and massive liver damage that culminated in death. In addition, we show that poly and monoclonal anti-DENV-specific antibodies can sufficiently increase viral replication through a suppression of early innate antiviral responses and enhance disease manifestation, so that a mostly non-lethal illness becomes a fatal disease resembling human DHF/DSS. Finally, treatment with intravenous immunoglobulin containing anti-DENV antibodies confirmed the potential enhancing capacity of subneutralizing antibodies to mediate virus infection and replication and induce severe disease manifestation of DENV-infected mice. Thus, our results show that humoral responses unleashed during DENV infections can exert protective or pathological outcomes and provide insight into the pathogenesis of this important human pathogen.

  12. Viral Load and Cytokine Response Profile Does Not Support Antibody-Dependent Enhancement in Dengue-Primed Zika Virus-Infected Patients.

    Science.gov (United States)

    Terzian, Ana Carolina Bernardes; Schanoski, Alessandra Soares; Mota, Mânlio Tasso de Oliveira; da Silva, Rafael Alves; Estofolete, Cássia Fernanda; Colombo, Tatiana Elias; Rahal, Paula; Hanley, Kathryn A; Vasilakis, Nikos; Kalil, Jorge; Nogueira, Maurício Lacerda

    2017-10-15

    The pathogenesis of severe dengue disease involves immune components as biomarkers. The mechanism by which some dengue virus (DENV)-infected individuals progress to severe disease is poorly understood. Most studies on the pathogenesis of severe dengue disease focus on the process of antibody-dependent enhancement (ADE) as a primary risk factor. With the circulation of Zika virus (ZIKV) in DENV-endemic areas, many people infected by ZIKV were likely exposed to DENV. The influence of such exposure on Zika disease outcomes remains unknown. We investigated whether patients previously exposed to DENV exhibited higher viremia when exposed to a subsequent, heterologous dengue or Zika infection than those patients not previously exposed to dengue. We measured viral loads and cytokine profile during patients' acute infections. Neither dengue nor Zika viremia was higher in patients with prior DENV infection, although the power to detect such a difference was only adequate in the ZIKV analysis. Of the 10 cytokines measured, only 1 significant difference was detected: Levels of interleukin 1β (IL-1β) were lower in dengue-infected patients who had experienced a previous dengue infection than patients infected with dengue for the first time. However, power to detect differences between groups was low. In Zika-infected patients, levels of IL-1β showed a significant, positive correlation with viral load. No signs of ADE were observed in vivo in patients with acute ZIKV infection who had prior exposure to DENV. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  13. Sulfated polysaccharide, curdlan sulfate, efficiently prevents entry/fusion and restricts antibody-dependent enhancement of dengue virus infection in vitro: a possible candidate for clinical application.

    Directory of Open Access Journals (Sweden)

    Koji Ichiyama

    Full Text Available Curdlan sulfate (CRDS, a sulfated 1→3-β-D glucan, previously shown to be a potent HIV entry inhibitor, is characterized in this study as a potent inhibitor of the Dengue virus (DENV. CRDS was identified by in silico blind docking studies to exhibit binding potential to the envelope (E protein of the DENV. CRDS was shown to inhibit the DENV replication very efficiently in different cells in vitro. Minimal effective concentration of CRDS was as low as 0.1 µg/mL in LLC-MK2 cells, and toxicity was observed only at concentrations over 10 mg/mL. CRDS can also inhibit DENV-1, 3, and 4 efficiently. CRDS did not inhibit the replication of DENV subgenomic replicon. Time of addition experiments demonstrated that the compound not only inhibited viral infection at the host cell binding step, but also at an early post-attachment step of entry (membrane fusion. The direct binding of CRDS to DENV was suggested by an evident reduction in the viral titers after interaction of the virus with CRDS following an ultrafiltration device separation, as well as after virus adsorption to an alkyl CRDS-coated membrane filter. The electron microscopic features also showed that CRDS interacted directly with the viral envelope, and caused changes to the viral surface. CRDS also potently inhibited DENV infection in DC-SIGN expressing cells as well as the antibody-dependent enhancement of DENV-2 infection. Based on these data, a probable binding model of CRDS to DENV E protein was constructed by a flexible receptor and ligand docking study. The binding site of CRDS was predicted to be at the interface between domains II and III of E protein dimer, which is unique to this compound, and is apparently different from the β-OG binding site. Since CRDS has already been tested in humans without serious side effects, its clinical application can be considered.

  14. A tetravalent virus-like particle vaccine designed to display domain III of dengue envelope proteins induces multi-serotype neutralizing antibodies in mice and macaques which confer protection against antibody dependent enhancement in AG129 mice.

    Directory of Open Access Journals (Sweden)

    Viswanathan Ramasamy

    2018-01-01

    Full Text Available Dengue is one of the fastest spreading vector-borne diseases, caused by four antigenically distinct dengue viruses (DENVs. Antibodies against DENVs are responsible for both protection as well as pathogenesis. A vaccine that is safe for and efficacious in all people irrespective of their age and domicile is still an unmet need. It is becoming increasingly apparent that vaccine design must eliminate epitopes implicated in the induction of infection-enhancing antibodies.We report a Pichia pastoris-expressed dengue immunogen, DSV4, based on DENV envelope protein domain III (EDIII, which contains well-characterized serotype-specific and cross-reactive epitopes. In natural infection, <10% of the total neutralizing antibody response is EDIII-directed. Yet, this is a functionally relevant domain which interacts with the host cell surface receptor. DSV4 was designed by in-frame fusion of EDIII of all four DENV serotypes and hepatitis B surface (S antigen and co-expressed with unfused S antigen to form mosaic virus-like particles (VLPs. These VLPs displayed EDIIIs of all four DENV serotypes based on probing with a battery of serotype-specific anti-EDIII monoclonal antibodies. The DSV4 VLPs were highly immunogenic, inducing potent and durable neutralizing antibodies against all four DENV serotypes encompassing multiple genotypes, in mice and macaques. DSV4-induced murine antibodies suppressed viremia in AG129 mice and conferred protection against lethal DENV-4 virus challenge. Further, neither murine nor macaque anti-DSV4 antibodies promoted mortality or inflammatory cytokine production when passively transferred and tested in an in vivo dengue disease enhancement model of AG129 mice.Directing the immune response to a non-immunodominant but functionally relevant serotype-specific dengue epitope of the four DENV serotypes, displayed on a VLP platform, can help minimize the risk of inducing disease-enhancing antibodies while eliciting effective tetravalent

  15. Assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing the antibody response against synthetic foot-and-mouth disease virus peptide

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yu-Shiun [Department of Materials Science and Engineering, National Chiao Tung University, 1001 University Road, EE137, Hsinchu 300, Taiwan (China); Hung, Yao-Ching [Department of Obstetrics and Gynecology, School of Medicine, China Medical University and Hospital, 91 Hsueh-Shih Road, Taichung 40402, Taiwan (China); Lin, Wei-Hsu [Institute of Nanotechnology, National Chiao Tung University, 1001 University Road, Hsinchu 300, Taiwan (China); Huang, Guewha Steven, E-mail: gstevehuang@mail.nctu.edu.tw [Department of Materials Science and Engineering, Institute of Nanotechnology, National Chiao Tung University, 1001 University Road, Hsinchu 300, Taiwan, Republic of China (China)

    2010-05-14

    To assess the ability of gold nanoparticles (GNPs) to act as a size-dependent carrier, a synthetic peptide resembling foot-and-mouth disease virus (FMDV) protein was conjugated to GNPs ranging from 2 to 50 nm in diameter (2, 5, 8, 12, 17, 37, and 50 nm). An extra cysteine was added to the C-terminus of the FMDV peptide (pFMDV) to ensure maximal conjugation to the GNPs, which have a high affinity for sulfhydryl groups. The resultant pFMDV-GNP conjugates were then injected into BALB/c mice. Immunization with pFMDV-keyhole limpet hemocyanin (pFMDV-KLH) conjugate was also performed as a control. Blood was obtained from the mice after 4, 6, 8, and 10 weeks and antibody titers against both pFMDV and the carriers were measured. For the pFMDV-GNP immunization, specific antibodies against the synthetic peptide were detected in the sera of mice injected with 2, 5, 8, 12, and 17 nm pFMDV-GNP conjugates. Maximal antibody binding was noted for GNPs of diameter 8-17 nm. The pFMDV-GNPs induced a three-fold increase in the antibody response compared to the response to pFMDV-KLH. However, sera from either immunized mouse group did not exhibit an antibody response to GNPs, while the sera from pFMDV-KLH-immunized mice presented high levels of binding activity against KLH. Additionally, the uptake of pFMDV-GNP in the spleen was examined by inductively coupled plasma mass spectroscopy (ICP-MS) and transmission electron microscopy (TEM). The quantity of GNPs that accumulated in the spleen correlated to the magnitude of the immune response induced by pFMDV-GNP. In conclusion, we demonstrated the size-dependent immunogenic properties of pFMDV-GNP conjugates. Furthermore, we established that GNPs ranging from 8 to 17 nm in diameter may be ideal for eliciting a focused antibody response against a synthetic pFMDV peptide.

  16. Dengue Virus-Specific Antibodies Enhance Brazilian Zika Virus Infection.

    Science.gov (United States)

    Castanha, Priscila M S; Nascimento, Eduardo J M; Braga, Cynthia; Cordeiro, Marli T; de Carvalho, Otávio V; de Mendonça, Leila R; Azevedo, Elisa A N; França, Rafael F O; Dhalia, Rafael; Marques, Ernesto T A

    2017-03-01

    Anti-Flavivirus antibodies are highly cross-reactive and may facilitate Zika virus (ZIKV) infection through the antibody-dependent enhancement (ADE) mechanism. We demonstrate that dengue-specific antibodies enhance the infection of a primary Brazilian ZIKV isolate in a FcγRII-expressing K562 cell line. In addition, we demonstrate that serum samples from dengue-immune pregnant women enhanced ZIKV infection. These findings highlight the need for epidemiological studies and animal models to further confirm the role of ADE in the development of congenital and neurological complications associated with ZIKV infections. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  17. Enhanced Phagocytosis and Antibody Production by Tinospora ...

    African Journals Online (AJOL)

    Tinospora cordifolia (guduchi) is a widely used shrub in ayurvedic systems of medicine known to possess immunomodulatory properties. In the present study the aqueous extract of T. cordifolia was found to enhance phagocytosis in vitro. The aqueous and ethanolic extracts also induced an increase in antibody production ...

  18. Epitope Mapping of Dengue-Virus-Enhancing Monoclonal-Antibody Using Phage Display Peptide Library

    OpenAIRE

    Chung-I Rai; Huan-Yao Lei; Yee-Shin Lin; Hsiao-Sheng Liu; Shun-Hua Chen; Lien-Cheng Chen; Trai-Ming Yeh

    2008-01-01

    The Antibody-Dependent Enhancement (ADE) hypothesis has been proposed to explain why more severe manifestations of Dengue Hemorrhagic Fever and Dengue Shock Syndrome (DHF/DSS) occur predominantly during secondary infections of Dengue Virus (DV) with different serotypes. However, the epitopes recognized by these enhancing antibodies are unclear. Recently, anti-pre-M monoclonal antibody (mAb 70-21), which recognized all DV serotypes without neutralizing activity, were generated and demonstrated...

  19. Zika Virus-Induced Antibody Response Enhances Dengue Virus Serotype 2 Replication In Vitro.

    Science.gov (United States)

    Kawiecki, Anna B; Christofferson, Rebecca C

    2016-11-01

    Zika virus has emerged in the Americas, where dengue virus is endemic. Among the 4 serotypes of dengue virus, antibody-dependent enhancement is thought to enhance viral replication and disease severity. Reports suggest that anti-dengue virus antibody may enhance Zika virus replication. We investigated whether Zika virus antibodies enhance dengue virus replication, by exposing C57Bl/6 mice to Zika virus. Polyclonal serum was verified for strong Zika virus-neutralizing, dengue virus-subneutralizing capacity. Then we determined the enhancement capabilities of Zika virus-immune serum for dengue virus in vitro. We showed that Zika virus antibodies have the ability to enhance dengue virus infections, which is important, because in many Zika virus-affected areas, dengue virus is expected to remain endemic. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  20. Anti-DNA antibody mediated catalysis is isotype dependent.

    Science.gov (United States)

    Xia, Yumin; Eryilmaz, Ertan; Zhang, Qiuting; Cowburn, David; Putterman, Chaim

    2016-01-01

    Anti-DNA antibodies are the serological hallmark of systemic lupus erythematosus, and participate in the pathogenesis of lupus nephritis by cross-reacting with multiple renal antigens. Previously, using a panel of murine anti-DNA IgGs that share identical variable regions but that differ in the constant regions, we demonstrated that the cross-reaction and renal pathogenicity of anti-DNA antibodies are isotype dependent. In this study, we investigated the catalytic potential of this anti-DNA antibody panel, and determined its isotype dependency. The three isotype switch variants (IgG1, IgG2a, IgG2b) and the parent IgG3 PL9-11 anti-DNA antibodies were compared in their catalysis of 500 base pair linear double stranded DNA and a 12-mer peptide (ALWPPNLHAWVP), by gel analysis, MALDI-TOF mass spectrometry, and nuclear magnetic resonance spectroscopy. The binding affinity of anti-DNA antibodies to double stranded DNA and peptide antigens were assessed by ELISA and surface plasmon resonance. We found that the PL9-11 antibody isotypes vary significantly in their potential to catalyze the cleavage of both linear and double stranded DNA and the proteolysis of peptides. The degree of the cleavage and proteolysis increases with the incubation temperature and time. While different PL9-11 isotypes have the same initial attack sites within the ALWPPNLHAWVP peptide, there was no correlation between binding affinity to the peptide and proteolysis rates. In conclusion, the catalytic properties of anti-DNA antibodies are isotype dependent. This finding provides further evidence that antibodies that share the same variable region, but which have different constant regions, are functionally distinct. The catalytic effects modulated by antibody constant regions need to be considered in the design of therapeutic antibodies (abzymes) and peptides designed to block pathogenic autoantibodies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Antibody-mediated enhancement of human immunodeficiency virus type 1 infectivity is determined by the structure of gp120 and depends on modulation of the gp120-CCR5 interaction

    NARCIS (Netherlands)

    C. Guillon (Christophe); M. Schutten (Martin); P.H.M. Boers (Patrick); R.A. Gruters (Rob); A.D.M.E. Osterhaus (Albert)

    2002-01-01

    textabstractIn this study, we characterized the viral determinants of coreceptor usage in relation to susceptibility to antibody-mediated neutralization or enhancement of infectivity by using chimeras of three highly related human immunodeficiency virus type 1 (HIV-1) isolates of different

  2. Dual antibody therapy to harness the innate anti-tumor immune response to enhance antibody targeting of tumors.

    Science.gov (United States)

    Chester, Cariad; Marabelle, Aurelien; Houot, Roch; Kohrt, Holbrook E

    2015-04-01

    Cancer immunotherapy is a rapidly evolving field that offers a novel paradigm for cancer treatment: therapies focus on enhancing the immune system's innate and adaptive anti-tumor response. Early immunotherapeutics have achieved impressive clinical outcomes and monoclonal antibodies are now integral to therapeutic strategies in a variety of cancers. However, only recently have antibodies targeting innate immune cells entered clinical development. Innate immune effector cells play important roles in generating and maintaining antitumor immunity. Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are important innate immune mechanisms for tumor eradication. These cytolytic processes are initiated by the detection of a tumor-targeting antibody and can be augmented by activating co-stimulatory pathways or blocking inhibitory signals on innate immune cells. The combination of FDA-approved monoclonal antibodies with innate effector-targeting antibodies has demonstrated potent preclinical therapeutic synergy and early-phase combinatorial clinical trials are ongoing. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Humanised IgG1 antibody variants targeting membrane-bound carcinoembryonic antigen by antibody-dependent cellular cytotoxicity and phagocytosis.

    Science.gov (United States)

    Ashraf, S Q; Umana, P; Mössner, E; Ntouroupi, T; Brünker, P; Schmidt, C; Wilding, J L; Mortensen, N J; Bodmer, W F

    2009-11-17

    The effect of glycoengineering a membrane specific anti-carcinoembryonic antigen (CEA) (this paper uses the original term CEA for the formally designated CEACAM5) antibody (PR1A3) on its ability to enhance killing of colorectal cancer (CRC) cell lines by human immune effector cells was assessed. In vivo efficacy of the antibody was also tested. The antibody was modified using EBNA cells cotransfected with beta-1,4-N-acetylglucosaminyltransferase III and the humanised hPR1A3 antibody genes. The resulting alteration of the Fc segment glycosylation pattern enhances the antibody's binding affinity to the FcgammaRIIIa receptor on human immune effector cells but does not alter the antibody's binding capacity. Antibody-dependent cellular cytotoxicity (ADCC) is inhibited in the presence of anti-FcgammaRIII blocking antibodies. This glycovariant of hPR1A3 enhances ADCC 10-fold relative to the parent unmodified antibody using either unfractionated peripheral blood mononuclear or natural killer (NK) cells and CEA-positive CRC cells as targets. NK cells are far more potent in eliciting ADCC than either freshly isolated monocytes or granulocytes. Flow cytometry and automated fluorescent microscopy have been used to show that both versions of hPR1A3 can induce antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages. However, the glycovariant antibody did not mediate enhanced ADCP. This may be explained by the relatively low expression of FcgammaRIIIa on cultured macrophages. In vivo studies show the efficacy of glycoengineered humanised IgG1 PR1A3 in significantly improving survival in a CRC metastatic murine model. The greatly enhanced in vitro ADCC activity of the glycoengineered version of hPR1A3 is likely to be clinically beneficial.

  4. FcγRII-binding Centyrins mediate agonism and antibody-dependent cellular phagocytosis when fused to an anti-OX40 antibody.

    Science.gov (United States)

    Zhang, Di; Whitaker, Brian; Derebe, Mehabaw G; Chiu, Mark L

    2018-04-01

    Immunostimulatory antibodies against the tumor necrosis factor receptors (TNFR) are emerging as promising cancer immunotherapies. The agonism activity of such antibodies depends on crosslinking to Fc gamma RIIB receptor (FcγRIIB) to enable the antibody multimerization that drives TNFR activation. Previously, Fc engineering was used to enhance the binding of such antibodies to Fcγ receptors. Here, we report the identification of Centyrins as alternative scaffold proteins with binding affinities to homologous FcγRIIB and FcγRIIA, but not to other types of Fcγ receptors. One Centyrin, S29, was engineered at distinct positions of an anti-OX40 SF2 antibody to generate bispecific and tetravalent molecules named as mAbtyrins. Regardless of the position of S29 on the SF2 antibody, SF2-S29 mAbtyrins could bind FcγRIIB and FcγRIIA specifically while maintaining binding to OX40 receptors. In a NFκB reporter assay, attachment of S29 Centyrin molecules at the C-termini, but not the N-termini, resulted in SF2 antibodies with increased agonism owing to FcγRIIB crosslinking. The mAbtyrins also showed agonism in T-cell activation assays with immobilized FcγRIIB and FcγRIIA, but this activity was confined to mAbtyrins with S29 specifically at the C-termini of antibody heavy chains. Furthermore, regardless of the position of the molecule, S29 Centyrin could equip an otherwise Fc-silent antibody with antibody-dependent cellular phagocytosis activity without affecting the antibody's intrinsic antibody-dependent cell-meditated cytotoxicity and complement-dependent cytotoxicity. In summary, the appropriate adoption FcγRII-binding Centyrins as functional modules represents a novel strategy to engineer therapeutic antibodies with improved functionalities.

  5. Antibody side chain conformations are position-dependent.

    Science.gov (United States)

    Leem, Jinwoo; Georges, Guy; Shi, Jiye; Deane, Charlotte M

    2018-01-10

    Side chain prediction is an integral component of computational antibody design and structure prediction. Current antibody modelling tools use backbone-dependent rotamer libraries with conformations taken from general proteins. Here we present our antibody-specific rotamer library, where rotamers are binned according to their immunogenetics (IMGT) position, rather than their local backbone geometry. We find that for some amino acid types at certain positions, only a restricted number of side chain conformations are ever observed. Using this information, we are able to reduce the breadth of the rotamer sampling space. Based on our rotamer library, we built a side chain predictor, position-dependent antibody rotamer swapper (PEARS). On a blind test set of 95 antibody model structures, PEARS had the highest average χ 1 and χ1+2 accuracy (78.7% and 64.8%) compared to three leading backbone-dependent side chain predictors. Our use of IMGT position, rather than backbone ϕ/ψ, meant that PEARS was more robust to errors in the backbone of the model structure. PEARS also achieved the lowest number of side chain-side chain clashes. PEARS is freely available as a web application at http://opig.stats.ox.ac.uk/webapps/pears. © 2018 Wiley Periodicals, Inc.

  6. Antibody-dependent cellular cytotoxicity in antimyelin antibody-induced oligodendrocyte damage in vitro.

    Science.gov (United States)

    Griot-Wenk, M; Griot, C; Pfister, H; Vandevelde, M

    1991-08-01

    Treatment of dissociated murine brain cell cultures with an antibody recognizing galactocerebroside (GalC) led to degeneration of oligodendrocytes with loss of their cell processes. F(ab')2 fragments prepared from this antibody showed no effect. The anti-GalC antibody--but not its F(ab')2 fragments b2 was able to stimulate macrophages in these cultures as seen in a chemiluminescence assay. Therefore, antibodies bound to oligodendrocytes stimulated nearby macrophages by interacting with their Fc receptors. The oligodendroglial damage coincided with the release of toxic compounds by the stimulated macrophages, since treatment of the cultures with the anti-GalC antibody and a variety of other macrophage stimulating agents led to secretion of reactive oxygen species and--in some experiments--tumor necrosis factor, both known to be toxic for oligodendrocytes. These in vitro experiments show evidence that antibody-dependent cellular cytotoxicity may be an important mechanism of tissue destruction in inflammatory demyelinating diseases.

  7. Augmentation of antibody-dependent cellular cytotoxicity with defucosylated monoclonal antibodies in patients with GI-tract cancer.

    Science.gov (United States)

    Nakajima, Takahiro; Okayama, Hirokazu; Ashizawa, Mai; Noda, Masaru; Aoto, Keita; Saito, Motonobu; Monma, Tomoyuki; Ohki, Shinji; Shibata, Masahiko; Takenoshita, Seiichi; Kono, Koji

    2018-02-01

    Enhancement of antibody-dependent cellular cytotoxicity (ADCC) with some modalities may be a promising approach to enhance the efficacy of therapeutic monoclonal antibodies (mAbs). It has previously been demonstrated that the removal of fucose from antibody oligosaccharides (defucosylation) leads to augmentation of ADCC activity. To establish clinically relevant evidence of this procedure, the present study evaluated trastuzumab- and cetuximab-mediated ADCC by comparing defucosylated mAbs with conventional mAbs using peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from 20 patients with gastrointestinal tract cancer and 10 healthy volunteers. ADCCs were measured using PBMCs as effector cells and two gastric cancer cell lines as target cells. ADCCs were significantly enhanced with defucosylated mAbs compared with conventional mAbs using PBMC from the healthy donors and patients with cancer. The results confirmed that the cetuximab- and trastuzumab-mediated ADCCs in advanced disease were impaired in comparison to those in early disease or healthy individuals. However, when the defucosylated mAbs were used instead of the conventional mAbs, the ADCC activities in the advanced cases were almost comparable with those in early disease or healthy individuals. Furthermore, the expression of ADCC associated molecules were modified toward immunosuppressive status with a mitogen-activated protein kinase inhibitor in vitro , the conventional cetuximab- and trastuzumab-mediated ADCC was downregulated, and the defucosylated mAbs overcome the downregulation of ADCC. In conclusion, defucosylated therapeutic mAbs may enhance ADCC activities in patients with cancer, which may lead to more effective anti-cancer treatments.

  8. CD47 limits antibody dependent phagocytosis against non-malignant B cells.

    Science.gov (United States)

    Gallagher, Sandra; Turman, Sean; Lekstrom, Kristen; Wilson, Susan; Herbst, Ronald; Wang, Yue

    2017-05-01

    Recent studies have demonstrated the importance of CD47 in protecting malignant B cells from antibody dependent cellular phagocytosis (ADCP). Combined treatment of anti-CD47 and -CD20 antibodies synergistically augment elimination of tumor B cells in xenograft mouse models. This has led to the development of novel reagents that can potentially enhance killing of malignant B cells in patients. B cell depleting therapy is also a promising treatment for autoimmune patients. In the current study, we aimed to investigate whether or not CD47 protects non-malignant B cells from ADCP. We show that CD47 is expressed on all B cells in mice, with the highest level on plasma cells in bone marrow and spleen. Although its expression is dispensable for B cell development in mice, CD47 on B cells limits antibody mediated phagocytosis. B cell depletion following in vivo anti-CD19 treatment is more efficient in CD47-/- mice than in wild type mice. In vitro, both naïve and activated B cells from CD47-/- mice are more sensitive to ADCP than wild type B cells. Lastly, we show in an ADCP assay that blocking CD47 can enhance anti-CD19 antibody mediated phagocytosis of wild type B cells. These results suggest that in addition to its already demonstrated benefit in cancer, targeting CD47 may be used as an adjunct in combination with B cell depletion antibodies for treatment of autoimmune diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Antibody dependent cellular phagocytosis by macrophages is a novel mechanism of action of elotuzumab.

    Science.gov (United States)

    Kurdi, Ahmed T; Glavey, Siobhan V; Bezman, Natalie A; Jhatakia, Amy; Guerriero, Jennifer L; Manier, Salomon; Moschetta, Michele; Mishima, Yuji; Roccaro, Aldo; Detappe, Alexandre; Liu, Chia-Jen; Sacco, Antonio; Huynh, Daisy; Tai, Yu-Tzu; Robbins, Michael D; Azzi, Jamil; Ghobrial, Irene M

    2018-04-13

    Elotuzumab, a recently approved antibody for the treatment of multiple myeloma (MM), has been shown to stimulate Fcγ receptor (FcγR)-mediated antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells towards myeloma cells. The modulatory effects of elotuzumab on other effector cells in the tumor microenvironment, however, has not been fully explored. Antibody dependent cellular phagocytosis (ADCP) is a mechanism by which macrophages contribute to anti-tumor potency of monoclonal antibodies. Herein, we studied the NK cell independent effect of elotuzumab on tumor associated macrophages (TAMs) using a xenograft tumor model deficient in NK and adaptive immune cells. We demonstrate significant anti-tumor efficacy of single agent elotuzumab in immunocompromised xenograft models of multiple myeloma, which is in part mediated by Fc-FcγR interaction of elotuzumab with macrophages. Elotuzumab is shown in this study to induce phenotypic activation of macrophages in-vivo and mediates ADCP of myeloma cells though a FcγR dependent manner in-vitro. Together, these findings propose a novel immune mediated mechanism by which elotuzumab exerts anti-myeloma activity and helps to provide rationale for combination therapies that can enhance macrophage activity. Copyright ©2018, American Association for Cancer Research.

  10. Antibody targeting of Cathepsin S induces antibody-dependent cellular cytotoxicity

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    Kwok Hang Fai

    2011-12-01

    Full Text Available Abstract Background Proteolytic enzymes have been implicated in driving tumor progression by means of their cancer cell microenvironment activity where they promote proliferation, differentiation, apoptosis, migration, and invasion. Therapeutic strategies have focused on attenuating their activity using small molecule inhibitors, but the association of proteases with the cell surface during cancer progression opens up the possibility of targeting these using antibody dependent cellular cytotoxicity (ADCC. Cathepsin S is a lysosomal cysteine protease that promotes the growth and invasion of tumour and endothelial cells during cancer progression. Our analysis of colorectal cancer patient biopsies shows that cathepsin S associates with the cell membrane indicating a potential for ADCC targeting. Results Here we report the cell surface characterization of cathepsin S and the development of a humanized antibody (Fsn0503h with immune effector function and a stable in vivo half-life of 274 hours. Cathepsin S is expressed on the surface of tumor cells representative of colorectal and pancreatic cancer (23%-79% positive expression. Furthermore the binding of Fsn0503h to surface associated cathepsin S results in natural killer (NK cell targeted tumor killing. In a colorectal cancer model Fsn0503h elicits a 22% cytotoxic effect. Conclusions This data highlights the potential to target cell surface associated enzymes, such as cathepsin S, as therapeutic targets using antibodies capable of elicitingADCC in tumor cells.

  11. Metal-Dependent Amyloid β-Degrading Catalytic Antibody Construct

    Science.gov (United States)

    Nishiyama, Yasuhiro; Taguchi, Hiroaki; Hara, Mariko; Planque, Stephanie A.; Mitsuda, Yukie; Paul, Sudhir

    2015-01-01

    Catalytic antibodies (catabodies) that degrade target antigens rapidly are rare. We describe the metal-dependence of catabody construct 2E6, an engineered heterodimer of immunoglobulin light chain variable domains that hydrolyzes amyloid β peptides (Aβ) specifically. In addition to the electrophilic phosphonate inhibitor of serine proteases, the metal chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline completely inhibited the hydrolysis of Aβ by catabody 2E6. Formation of catabody-electrophilic phosphonate inhibitor adducts was unaffected by EDTA, suggesting that the metal exerts a favorable effect on a catalytic step after the initial catabody nucleophilic attack on Aβ. The EDTA inactivated catabody failed to disaggregate fibrillar Aβ, indicating the functional importance of the Aβ hydrolytic activity. Treating the EDTA-inactivated catabody with Zn2+ or Co2+ restored the Aβ hydrolytic activity, and Zn2+-induced catabody conformational transitions were evident by fluorescence emission spectroscopy. The studies reveal the absolute catabody dependence on a metal cofactor. PMID:24698848

  12. Human Cytomegalovirus Infection Increases Both Antibody- and Non–Antibody-Dependent Cellular Reactivity by Natural Killer Cells

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    Clive M. Michelo, PhD

    2017-12-01

    Conclusions. With regard to organ transplantation, these data suggest that CMV infection enhances NK cell alloreactivity, which may pose an additional adverse effect on graft survival, especially in the presence of donor specific antibodies.

  13. Naturally Acquired Human Immunity to Pneumococcus Is Dependent on Antibody to Protein Antigens.

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    Robert Wilson

    2017-01-01

    Full Text Available Naturally acquired immunity against invasive pneumococcal disease (IPD is thought to be dependent on anti-capsular antibody. However nasopharyngeal colonisation by Streptococcus pneumoniae also induces antibody to protein antigens that could be protective. We have used human intravenous immunoglobulin preparation (IVIG, representing natural IgG responses to S. pneumoniae, to identify the classes of antigens that are functionally relevant for immunity to IPD. IgG in IVIG recognised capsular antigen and multiple S. pneumoniae protein antigens, with highly conserved patterns between different geographical sources of pooled human IgG. Incubation of S. pneumoniae in IVIG resulted in IgG binding to the bacteria, formation of bacterial aggregates, and enhanced phagocytosis even for unencapsulated S. pneumoniae strains, demonstrating the capsule was unlikely to be the dominant protective antigen. IgG binding to S. pneumoniae incubated in IVIG was reduced after partial chemical or genetic removal of bacterial surface proteins, and increased against a Streptococcus mitis strain expressing the S. pneumoniae protein PspC. In contrast, depletion of type-specific capsular antibody from IVIG did not affect IgG binding, opsonophagocytosis, or protection by passive vaccination against IPD in murine models. These results demonstrate that naturally acquired protection against IPD largely depends on antibody to protein antigens rather than the capsule.

  14. Involvement of gamma interferon in antibody enhancement by adjuvants.

    Science.gov (United States)

    Odean, M J; Frane, C M; Van derVieren, M; Tomai, M A; Johnson, A G

    1990-02-01

    In a previous study the adjuvant action of a monophosphoryl lipid A, a nontoxic derivative of endotoxic lipopolysaccharide (LPS), was found to be negated by a monoclonal anti-gamma interferon (anti-IFN-gamma) antibody. The present investigation centered on three other adjuvants of diverse microbial origins, testing for their capacity to affect the release of IFN-gamma as an explanation for their antibody-enhancing action. The adjuvant action of each of the three, a wild-type LPS, synthetic poly(A)-poly(U) complexes, and a synthetic muramyl dipeptide, n-acetylmuramyl-L-alanyl-D-glutaminyl-n-butyl ester (murabutide), was transferable by adjuvant-stimulated T cells to normal spleen cells on coculture. Supernatant fluids from these T cells contained increased levels of IFN-gamma. Addition of a monoclonal anti-IFN-gamma antibody to adjuvant-stimulated spleen cell cultures reduced the adjuvant action by approximately one-half. Removal of natural killer cells from spleen cell populations prior to culture with antigen had no effect on the enhancement induced by LPS and monophosphoryl lipid A. It was concluded that the enhancement induced by the adjuvants LPS, poly(A)-poly(U), and murabutide is mediated in part by their action on T cells resulting in release of IFN-gamma suggesting activation of a common transmembrane signal.

  15. Dengue viruses are enhanced by distinct populations of serotype cross-reactive antibodies in human immune sera.

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    Ruklanthi de Alwis

    2014-10-01

    Full Text Available Dengue viruses (DENV are mosquito-borne flaviviruses of global importance. DENV exist as four serotypes, DENV1-DENV4. Following a primary infection, individuals produce DENV-specific antibodies that bind only to the serotype of infection and other antibodies that cross-react with two or more serotypes. People exposed to a secondary DENV infection with another serotype are at greater risk of developing more severe forms of dengue disease. The increased risk of severe dengue in people experiencing repeat DENV infections appear to be due, at least in part, to the ability of pre-existing serotype cross-reactive antibodies to form virus-antibody complexes that can productively infect Fcγ receptor-bearing target cells. While the theory of antibody-dependent enhancement (ADE is supported by several human and small animal model studies, the specific viral antigens and epitopes recognized by enhancing human antibodies after natural infections have not been fully defined. We used antibody-depletion techniques to remove DENV-specific antibody sub-populations from primary DENV-immune human sera. The effects of removing specific antibody populations on ADE were tested both in vitro using K562 cells and in vivo using the AG129 mouse model. Removal of serotype cross-reactive antibodies ablated enhancement of heterotypic virus infection in vitro and antibody-enhanced mortality in vivo. Further depletion studies using recombinant viral antigens showed that although the removal of DENV E-specific antibodies using recombinant E (rE protein resulted in a partial reduction in DENV enhancement, there was a significant residual enhancement remaining. Competition ADE studies using prM-specific Fab fragments in human immune sera showed that both rE-specific and prM-specific antibodies in primary DENV-immune sera significantly contribute to enhancement of heterotypic DENV infection in vitro. Identification of the targets of DENV-enhancing antibodies should contribute to

  16. Comparison of Antibody-Dependent Cell-Mediated Cytotoxicity and Virus Neutralization by HIV-1 Env-Specific Monoclonal Antibodies.

    Science.gov (United States)

    von Bredow, Benjamin; Arias, Juan F; Heyer, Lisa N; Moldt, Brian; Le, Khoa; Robinson, James E; Zolla-Pazner, Susan; Burton, Dennis R; Evans, David T

    2016-07-01

    Although antibodies to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein have been studied extensively for their ability to block viral infectivity, little data are currently available on nonneutralizing functions of these antibodies, such as their ability to eliminate virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 Env-specific antibodies of diverse specificities, including potent broadly neutralizing and nonneutralizing antibodies, were therefore tested for ADCC against cells infected with a lab-adapted HIV-1 isolate (HIV-1NL4-3), a primary HIV-1 isolate (HIV-1JR-FL), and a simian-human immunodeficiency virus (SHIV) adapted for pathogenic infection of rhesus macaques (SHIVAD8-EO). In accordance with the sensitivity of these viruses to neutralization, HIV-1NL4-3-infected cells were considerably more sensitive to ADCC, both in terms of the number of antibodies and magnitude of responses, than cells infected with HIV-1JR-FL or SHIVAD8-EO ADCC activity generally correlated with antibody binding to Env on the surfaces of virus-infected cells and with viral neutralization; however, neutralization was not always predictive of ADCC, as instances of ADCC in the absence of detectable neutralization, and vice versa, were observed. These results reveal incomplete overlap in the specificities of antibodies that mediate these antiviral activities and provide insights into the relationship between ADCC and neutralization important for the development of antibody-based vaccines and therapies for combating HIV-1 infection. This study provides fundamental insights into the relationship between antibody-dependent cell-mediated cytotoxicity (ADCC) and virus neutralization that may help to guide the development of antibody-based vaccines and immunotherapies for the prevention and treatment of HIV-1 infection. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  17. Antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus.

    Science.gov (United States)

    Yip, Ming Shum; Leung, Nancy Hiu Lan; Cheung, Chung Yan; Li, Ping Hung; Lee, Horace Hok Yeung; Daëron, Marc; Peiris, Joseph Sriyal Malik; Bruzzone, Roberto; Jaume, Martial

    2014-05-06

    Public health risks associated to infection by human coronaviruses remain considerable and vaccination is a key option for preventing the resurgence of severe acute respiratory syndrome coronavirus (SARS-CoV). We have previously reported that antibodies elicited by a SARS-CoV vaccine candidate based on recombinant, full-length SARS-CoV Spike-protein trimers, trigger infection of immune cell lines. These observations prompted us to investigate the molecular mechanisms and responses to antibody-mediated infection in human macrophages. We have used primary human immune cells to evaluate their susceptibility to infection by SARS-CoV in the presence of anti-Spike antibodies. Fluorescence microscopy and real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) were utilized to assess occurrence and consequences of infection. To gain insight into the underlying molecular mechanism, we performed mutational analysis with a series of truncated and chimeric constructs of fragment crystallizable γ receptors (FcγR), which bind antibody-coated pathogens. We show here that anti-Spike immune serum increased infection of human monocyte-derived macrophages by replication-competent SARS-CoV as well as Spike-pseudotyped lentiviral particles (SARS-CoVpp). Macrophages infected with SARS-CoV, however, did not support productive replication of the virus. Purified anti-viral IgGs, but not other soluble factor(s) from heat-inactivated mouse immune serum, were sufficient to enhance infection. Antibody-mediated infection was dependent on signaling-competent members of the human FcγRII family, which were shown to confer susceptibility to otherwise naïve ST486 cells, as binding of immune complexes to cell surface FcγRII was necessary but not sufficient to trigger antibody-dependent enhancement (ADE) of infection. Furthermore, only FcγRII with intact cytoplasmic signaling domains were competent to sustain ADE of SARS-CoVpp infection, thus providing additional

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

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    Milly M Choy

    2015-11-01

    Full Text Available 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.

  19. Microglial internalization and degradation of pathological tau is enhanced by an anti-tau monoclonal antibody.

    Science.gov (United States)

    Luo, Wenjie; Liu, Wencheng; Hu, Xiaoyan; Hanna, Mary; Caravaca, April; Paul, Steven M

    2015-06-09

    Microglia have been shown to contribute to the clearance of brain amyloid β peptides (Aβ), the major component of amyloid plaques, in Alzheimer's disease (AD). However, it is not known whether microglia play a similar role in the clearance of tau, the major component of neurofibrillary tangles (NFTs). We now report that murine microglia rapidly internalize and degrade hyperphosphorylated pathological tau isolated from AD brain tissue in a time-dependent manner in vitro. We further demonstrate that microglia readily degrade human tau species released from AD brain sections and eliminate NFTs from brain sections of P301S tauopathy mice. The anti-tau monoclonal antibody MC1 enhances microglia-mediated tau degradation in an Fc-dependent manner. Our data identify a potential role for microglia in the degradation and clearance of pathological tau species in brain and provide a mechanism explaining the potential therapeutic actions of passively administered anti-tau monoclonal antibodies.

  20. Enhanced neutralization potency of botulinum neurotoxin antibodies using a red blood cell-targeting fusion protein.

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    Sharad P Adekar

    2011-03-01

    Full Text Available Botulinum neurotoxin (BoNT potently inhibits cholinergic signaling at the neuromuscular junction. The ideal countermeasures for BoNT exposure are monoclonal antibodies or BoNT antisera, which form BoNT-containing immune complexes that are rapidly cleared from the general circulation. Clearance of opsonized toxins may involve complement receptor-mediated immunoadherence to red blood cells (RBC in primates or to platelets in rodents. Methods of enhancing immunoadherence of BoNT-specific antibodies may increase their potency in vivo. We designed a novel fusion protein (FP to link biotinylated molecules to glycophorin A (GPA on the RBC surface. The FP consists of an scFv specific for murine GPA fused to streptavidin. FP:mAb:BoNT complexes bound specifically to the RBC surface in vitro. In a mouse model of BoNT neutralization, the FP increased the potency of single and double antibody combinations in BoNT neutralization. A combination of two antibodies with the FP gave complete neutralization of 5,000 LD50 BoNT in mice. Neutralization in vivo was dependent on biotinylation of both antibodies and correlated with a reduction of plasma BoNT levels. In a post-exposure model of intoxication, FP:mAb complexes gave complete protection from a lethal BoNT/A1 dose when administered within 2 hours of toxin exposure. In a pre-exposure prophylaxis model, mice were fully protected for 72 hours following administration of the FP:mAb complex. These results demonstrate that RBC-targeted immunoadherence through the FP is a potent enhancer of BoNT neutralization by antibodies in vivo.

  1. Separation of cell-dependent antibody (CDA) and inhibitory antibody by protein-A affinity chromatography and the effect of fractions on antibody-dependent cellular cytotoxicity (ADCC).

    Science.gov (United States)

    Sato, N; Yabuki, Y; Toh, K; Ishii, Y; Kikuchi, K

    1979-01-01

    The nature of cell-dependent antibody (CDA) and the mechanism of inhibition of antibody-dependent cellular cytotoxicity (ADCC) were studied in the ADCC assay system in which culture cells of methylcholanthrene-induced rat fibrosarcoma (KMT-50) were used as target cells, xenogeneic antiserum (rabbit anti-KMT-50) as the CDA, and human peripheral blood leucocytes (PBL) as effector cells, respectively. By using protein-A Sepharose CL-4B affinity column chromatography of rabbit anti-KMT-50 serum, CDA was shown to bind protein A. Complement dependent-cytotoxicity (CDC), however, was demonstrated in both the adsorbed fraction (eluate) and the non-adsorbed fraction (effluent) to protein A from the same affinity column chromatography. These data confirmed that CDA was IgG with an intact Fc portion. Inhibition of ADCC occurred by pretreatment of effector cells with rabbit anti-effector (human PBL) serum even with extremely small amounts of antiserum. Such inhibition was demonstrated with the eluate but not with the effluent from protein-A Sepharose CL-4B affinity column chromatography of rabbit anti-effector serum. F(ab')2 fragments of the same eluate (IgG) did not inhibit the ADCC activity. These data showed that the inhibition of ADCC was induced by the blocking of Fc receptors of effector cells with the Fc portions of IgG in anti-effector serum. The data obtained indicate the usefulness of protein A in separation and analysis of CDA and in investigation of the inhibitory mechanisms of ADCC. Images Figure 2 PMID:437836

  2. Natural killer (NK cell mediated antibody-dependent cellular cytotoxicity (ADCC in tumour immunotherapy with therapeutic antibodies

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    Ursula Jördis Eva Seidel

    2013-03-01

    Full Text Available In the last decade several therapeutic antibodies have been FDA and EMEA approved. Although their mechanisms of action in vivo is not fully elucidated, antibody-dependent cellular cytotoxicity (ADCC mediated by natural killer (NK cells is presumed to be a key effector function. A substantial role of ADCC has been demonstrated in vitro and in mouse tumour models. However, a direct in vivo effect of ADCC in tumour reactivity in humans remains to be shown. Several studies revealed a predictive value of FcγRIIIa-V158F polymorphism in monoclonal antibody treatment, indicating a potential effect of ADCC on outcome for certain indications. Furthermore, the use of therapeutic antibodies after allogeneic haematopoietic stem cell transplantation is an interesting option. Studying the role of the FcγRIIIa-V158F polymorphism and the influence of KIR-receptor-ligand incompatibility on ADCC in this approach may contribute to future transplantation strategies. Despite the success of approved second-generation antibodies in the treatment of several malignancies, efforts are made to further augment ADCC in vivo by antibody engineering. Here, we review currently used therapeutic antibodies for which ADCC has been suggested as effector function.

  3. Characterization of Antibody-Dependent Killing of Trypanosomes by Macrophages.

    Science.gov (United States)

    1983-04-01

    werse e f08~m~ id’ Vm@ Trpnsm rhodesiense; trypanosomes; African trypanosomiasis ; protozoa, jpiiii tes macrophages; phagocytosis; antibody SEE...agglutinating antibody in the 19H im oglobulin fraction of rabbit antistrum during eaxwtntal African trypanosomiasis . .:283-292. Silverstein, S.C., R.N...species specificity in the action of complement since dog, human , guinea pig and mouse sera could be used as complement sources.4 In the presence of

  4. Intraperitoneal delivery of monoclonal antibodies: enhanced regional delivery advantage using intravenous unlabeled anti-mouse antibody

    International Nuclear Information System (INIS)

    Wahl, R.L.; Fisher, S.

    1987-01-01

    Radiolabeled monoclonal antibodies (MAb) delivered intraperitoneally expose cells in contact with peritoneal fluid to considerably higher levels of MAb than if the MAb dose were given intravenously. This regional delivery advantage for intact MAb is present mainly due to the relatively slow exit of MAb from the peritoneal fluid to the blood. Eventually, following i.p. injection, blood levels of MAb rise resulting in exposure of the animal to high systemic MAb levels and potential toxicity. In this series of experiments, systemic exposure was minimized by the administration of unlabeled goat polyclonal anti-mouse antibody intravenously from 1 1/2 to 6 h following i.p. MAb injection. This maneuver results in the formation of immune complexes with their subsequent clearance and dehalogenation by the reticuloendothelial system, thus minimizing systemic MAb exposure. This approach, of increasing systemic clearance of MAb, did not alter intraperitoneal MAb levels and thus significantly increased the regional delivery advantage to the peritoneal cavity by 70-100%. This approach provides an immunologic rationale for the further enhancement of MAb delivery to i.p. foci of malignant disease and may have diagnostic and therapeutic utility. (author)

  5. Lymphocyte antibody-dependent cytotoxicity test for evaluation of clinical role of monoclonal anti-D-antibodies for prevention of rhesus sensitization.

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    Olovnikova, N I; Belkina, E V; Nikolaeva, T L; Miterev, G Yu; Chertkov, I L

    2006-01-01

    Monoclonal antibodies to D antigen were studied in the reaction of antibody-dependent cytotoxicity for evaluation of the possibility of using these antibodies for preventing rhesus sensitization. High hemolytic activity of four anti-D-monoclonal antibodies in the antibody-dependent cytotoxicity test, mediated by their interaction with FcgammaRI, and the capacity to accelerate elimination of D+ erythrocytes from circulation did not provide the immunosuppressive effect. It was hypothesized that monoclonal antibodies for prevention of rhesus sensitization should interact with FcgammaRIII on lymphocytes. These monoclonal antibodies are extremely rare: only 4 of 125 studied antibodies mediated hemolysis in the antibody-dependent cytotoxicity test with lymphocytes, while all polyclonal anti-D-preparations exhibited this activity.

  6. Transmembrane TNF-dependent uptake of anti-TNF antibodies.

    Science.gov (United States)

    Deora, Arun; Hegde, Subramanya; Lee, Jacqueline; Choi, Chee-Ho; Chang, Qing; Lee, Cheryl; Eaton, Lucia; Tang, Hua; Wang, Dongdong; Lee, David; Michalak, Mark; Tomlinson, Medha; Tao, Qingfeng; Gaur, Nidhi; Harvey, Bohdan; McLoughlin, Shaun; Labkovsky, Boris; Ghayur, Tariq

    TNF-α (TNF), a pro-inflammatory cytokine is synthesized as a 26 kDa protein, anchors in the plasma membrane as transmembrane TNF (TmTNF), and is subjected to proteolysis by the TNF-α converting enzyme (TACE) to release the 15 kDa form of soluble TNF (sTNF). TmTNF and sTNF interact with 2 distinct receptors, TNF-R1 (p55) and TNF-R2 (p75), to mediate the multiple biologic effects of TNF described to date. Several anti-TNF biologics that bind to both forms of TNF and block their interactions with the TNF receptors are now approved for the treatment of a variety of immune-mediated diseases. Several reports suggest that binding of anti-TNFs to TmTNF delivers an outside-to-inside 'reverse' signal that may also contribute to the efficacy of anti-TNFs. Some patients, however, develop anti-TNF drug antibody responses (ADA or immunogenicity). Here, we demonstrate biochemically that TmTNF is transiently expressed on the surface of lipopolysaccharide-stimulated primary human monocytes, macrophages, and monocyte-derived dendritic cells (DCs) and expression of TmTNF on the cell surface is enhanced following treatment of cells with TAPI-2, a TACE inhibitor. Importantly, binding of anti-TNFs to TmTNF on DCs results in rapid internalization of the anti-TNF/TmTNF complex first into early endosomes and then lysosomes. The internalized anti-TNF is processed and anti-TNF peptides can be eluted from the surface of DCs. Finally, tetanus toxin peptides fused to anti-TNFs are presented by DCs to initiate T cell recall proliferation response. Collectively, these observations may provide new insights into understanding the biology of TmTNF, mode of action of anti-TNFs, biology of ADA response to anti-TNFs, and may help with the design of the next generation of anti-TNFs.

  7. Antibody dependent cellular phagocytosis (ADCP) and antibody dependent cellular cytotoxicity (ADCC) of breast cancer cells mediated by bispecific antibody, MDX-210.

    Science.gov (United States)

    Watanabe, M; Wallace, P K; Keler, T; Deo, Y M; Akewanlop, C; Hayes, D F

    1999-02-01

    MDX-210 is a bispecific antibody (BsAb) with specificity for both the proto-oncogene product of HER-2/neu (c-erbB-2) and FcgammaRI (CD64). HER-2/neu is overexpressed in malignant tissue of approximately 30% of patients with breast cancer, and FcgammaRI is expressed on human monocytes, macrophages, and IFN-gamma activated granulocytes. We investigated phagocytosis and cytolysis of cultured human breast cancer cells by human monocyte-derived macrophages (MDM) mediated by BsAb MDX-210, its partially humanized derivative (MDX-H210), and its parent MoAb 520C9 (anti-HER-2/neu) under various conditions. Purified monocytes were cultured with GM-CSF, M-CSF, or no cytokine for five or six days. Antibody dependent cellular phagocytosis (ADCP) and cytolysis (ADCC) assays were performed with the MDM and HER-2/neu positive target cells (SK-BR-3). ADCP was measured by two-color fluorescence flow cytometry using PKH2 (green fluorescent dye) and phycoerythrin-conjugated (red) monoclonal antibodies (MoAb) against human CD14 and CD11b. ADCC was measured with a non-radioactive LDH detection kit. Both BsAb MDX-210 (via FcgammaRI) and MoAb 520C9 (mouse IgG1, via FcgammaRII) mediated similar levels of ADCP and ADCC. ADCP mediated by BsAb MDX-H210 was identical to that mediated by BsAb MDX-210. Confocal microscopy demonstrated that dual-labeled cells represented true phagocytosis. Both ADCP and ADCC were higher when MDM were pre-incubated with GM-CSF than when incubated with M-CSF. BsAb MDX-210 is as active in vitro as the parent MoAb 520C9 in inducing both phagocytosis and cytolysis of MDM. MDX-210 and its partially humanized derivative, MDX-H210, mediated similar levels of ADCP. GM-CSF appears to superior to M-CSF in inducing MDM-mediated ADCC and ADCP. These studies support the ongoing clinical investigations of BsAb MDX-210 and its partially humanized derivative.

  8. Hydrogel Tethering Enhances Interdomain Stabilization of Single-Chain Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Yijia [Department; Ford, Nicole R. [Marine; Hecht, Karen A. [Marine; Roesijadi, Guritno [Marine; Department; Squier, Thomas C. [Department

    2017-10-12

    Self-assembly of recombinant proteins within the biosilica of living diatoms represents a means to construct functional materials in a reproducible and scalable manner that enable applications that harness the inherent specificities of proteins to sense and respond to environmental cues. Here we describe the use of a silaffin-derived lysine-rich 39 amino-acid targeting sequence (Sil3T8) to direct a single chain fragment variable (scFv) antibody or an enhanced green fluorescent protein (EGFP) to assemble within the biosilica frustule, resulting in abundances in excess of 200,000 proteins per frustule. The fluorescence of either a derivative of trinitrotoluene (TNT) bound to the scFv or the endogenous fluorescence of EGFP was used to monitor pro-tein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability. We find that proteins within isolated frustules undergo isotropic rotational motions with two-fold increases in rotational correlation times, which are indicative of weak macromolecular associations within the biosilica. Solvent accessibilities and high-affinity (pM) binding are comparable to those in solution. In contrast to solution conditions, scFv antibod-ies within the biosilica matrix retain their binding affinity in the presence of chaotropic agents (i.e., 8 M urea). These results argue that dramatic increases in protein conforma-tional stability within the biosilica frustule matrices arise through molecular crowding, acting to retain native protein folds and associated functionality to allow the utility of engineered proteins under a range of harsh environmental conditions associated with environmental sensing and industrial catalytic transformations.

  9. Dependability enhancing mechanisms for integrated clinical environments.

    Science.gov (United States)

    Zhao, Wenbing; Yang, Mary Q

    2017-10-01

    In this article, we present a set of lightweight mechanisms to enhance the dependability of a safety-critical real-time distributed system referred to as an integrated clinical environment (ICE). In an ICE, medical devices are interconnected and work together with the help of a supervisory computer system to enhance patient safety during clinical operations. Inevitably, there are strong dependability requirements on the ICE. We introduce a set of mechanisms that essentially make the supervisor component a trusted computing base, which can withstand common hardware failures and malicious attacks. The mechanisms rely on the replication of the supervisor component and employ only one input-exchange phase into the critical path of the operation of the ICE. Our analysis shows that the runtime latency overhead is much lower than that of traditional approaches.

  10. Pseudothrombocytopenia: an immunologic study on platelet antibodies dependent on ethylene diamine tetra-acetate

    NARCIS (Netherlands)

    Pegels, J. G.; Bruynes, E. C.; Engelfriet, C. P.; von dem Borne, A. E.

    1982-01-01

    Antibodies specifically reacting with platelets only in the presence of EDTA, by the platelet immunofluorescence test, were found in the serum of 20 patients with pseudothrombocytopenia due to in vitro EDTA-dependent platelet agglutination. These antibodies reacted optimally at 0-4 degree C. In 19

  11. Naturally Acquired Human Immunity to Pneumococcus Is Dependent on Antibody to Protein Antigens

    NARCIS (Netherlands)

    Wilson, R. (Robert); J. Cohen (Jonathan); Reglinski, M. (Mark); R.J. Jose; Chan, W.Y. (Win Yan); Marshall, H. (Helina); C.P. de Vogel (Corné); S.B. Gordon (Stephen); Goldblatt, D. (David); Petersen, F.C. (Fernanda C.); H. Baxendale (Helen); Brown, J.S. (Jeremy S.)

    2017-01-01

    textabstractNaturally acquired immunity against invasive pneumococcal disease (IPD) is thought to be dependent on anti-capsular antibody. However nasopharyngeal colonisation by Streptococcus pneumoniae also induces antibody to protein antigens that could be protective. We have used human intravenous

  12. Comparison of infection-neutralizing and -enhancing antibody balance induced by two distinct genotype strains of dengue virus type 1 or 3 DNA vaccines in mice.

    Science.gov (United States)

    Sjatha, Fithriyah; Takizawa, Yamato; Kotaki, Tomohiro; Yamanaka, Atsushi; Konishi, Eiji

    2013-11-01

    Dengue viruses have spread throughout tropical and subtropical countries, and vaccine development is urgently needed. However, one concern is that induction of insufficient levels of neutralizing antibodies in vaccines may increase disease severity because of a hypothetical mechanism termed antibody-dependent enhancement of infection. This study used two distinct genotype strains of dengue virus types 1 and 3 (DENV1 and DENV3, respectively) to compare antibody responses in a mouse-DNA vaccine model. As expected, a conventional neutralization test using Vero cells showed higher antibody titers in homologous rather than heterologous combinations of genotype strains used for mouse immunization and the neutralization test, for each of DENV1 and DENV3. However, our assay system using K562 cells to measure the balance of neutralizing and enhancing antibodies indicated that Vero cell-neutralizing antibody titers did not always correlate with enhancing activities observed at subneutralizing doses. Rather, induction of enhancing activities depended on the genotype strain used for mouse immunization. The genotype/strain difference also affected IgG subclass profiles and potentially the composition of antibody species induced in mice. This study suggests that enhancing activities of dengue virus-induced neutralizing antibodies may vary according to the genotype and has implications for vaccine antigen development. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  13. Characterization of Antibody-Dependent Killing of Trypanosomes by Macrophages.

    Science.gov (United States)

    1985-01-01

    specific antibody, murine peritoneal macrophages and macrophage-like cell lines bind Trypanosoma rhodesiense parasites in vitro. Using a large bank of anti ...as T. cruzi are able to actively penetrate host cells. However, the mechanism of cellular uptake of T. rhodesiense is not known. We therefore...intracellular parasites. Am. J. Trop. Med. Hyg. 26:161. 18. Takayanagi, T. and Y. Nakatake. 1977. Trypanosoma fambiense: The binding activity of antiserum to

  14. Oral antibiotics enhance antibody responses to keyhole limpet hemocyanin in orally but not muscularly immunized chickens.

    Science.gov (United States)

    Murai, Atsushi; Kitahara, Kazuki; Okumura, Shouta; Kobayashi, Misato; Horio, Fumihiko

    2016-02-01

    Recent studies have emphasized the crucial role of gut microbiota in triggering and modulating immune response. We aimed to determine whether the modification of gut microbiota by oral co-administration of two antibiotics, ampicillin and neomycin, would lead to changes in the antibody response to antigens in chickens. Neonatal chickens were given or not given ampicillin and neomycin (0.25 and 0.5 g/L, respectively) in drinking water. At 2 weeks of age, the chicks were muscularly or orally immunized with antigenic keyhole limpet hemocyanin (KLH), and then serum anti-KLH antibody levels were examined by ELISA. In orally immunized chicks, oral antibiotics treatment enhanced antibody responses (IgM, IgA, IgY) by 2-3-fold compared with the antibiotics-free control, while the antibiotics did not enhance antibody responses in the muscularly immunized chicks. Concomitant with their enhancement of antibody responses, the oral antibiotics also lowered the Lactobacillus species in feces. Low doses of antibiotics (10-fold and 100-fold lower than the initial trial), which failed to change the fecal Lactobacillus population, did not modify any antibody responses when chicks were orally immunized with KLH. In conclusion, oral antibiotics treatment enhanced the antibody response to orally exposed antigens in chickens. This enhancement of antibody response was associated with a modification of the fecal Lactobacillus content, suggesting a possible link between gut microbiota and antibody response in chickens. © 2015 Japanese Society of Animal Science.

  15. Antigenic specificity of antibody-dependent cell-mediated cytotoxicity directed against human immunodeficiency virus in antibody-positive sera.

    OpenAIRE

    Koup, R A; Sullivan, J L; Levine, P H; Brewster, F; Mahr, A; Mazzara, G; McKenzie, S; Panicali, D

    1989-01-01

    Antibody-dependent cell-mediated cytotoxicity (ADCC) specific for human immunodeficiency virus (HIV) has been described for HIV-infected individuals. To determine the antigenic specificity of this immune response and to define its relationship to the disease state, an ADCC assay was developed using Epstein-Barr virus-transformed lymphoblastoid cell line targets infected with vaccinia virus vectors expressing HIV proteins. The vaccinia virus vectors induced appropriate HIV proteins (envelope g...

  16. Enzymatic Inactivation of Endogenous IgG by IdeS Enhances Therapeutic Antibody Efficacy.

    Science.gov (United States)

    Järnum, Sofia; Runström, Anna; Bockermann, Robert; Winstedt, Lena; Crispin, Max; Kjellman, Christian

    2017-09-01

    Endogenous plasma IgG sets an immunologic threshold that dictates the activity of tumor-directed therapeutic antibodies. Saturation of cellular antibody receptors by endogenous antibody limits antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Here, we show how enzymatic cleavage of IgG using the bacterial enzyme IdeS can be utilized to empty both high and low affinity Fcγ-receptors and clear the entire endogenous antibody pool. Using in vitro models, tumor animal models as well as ex vivo analysis of sera collected during a previous clinical trial with IdeS, we show how clearing of competing plasma antibody levels with IdeS unblocks cellular antibody receptors. We show that therapeutic antibodies against breast cancer (trastuzumab), colon cancer (cetuximab), and lymphomas (rituximab and alemtuzumab) can be potentiated when endogenous IgG is removed. Overall, IdeS is shown to be a potent tool to reboot the human antibody repertoire and to generate a window to preferentially load therapeutic antibodies onto effector cells and thereby create an armada of dedicated tumor-seeking immune cells. Mol Cancer Ther; 16(9); 1887-97. ©2017 AACR . ©2017 American Association for Cancer Research.

  17. Antibody-nanoparticle conjugates to enhance the sensitivity of ELISA-based detection methods.

    Directory of Open Access Journals (Sweden)

    Margaret M Billingsley

    Full Text Available Accurate antigen detection is imperative for clinicians to diagnose disease, assess treatment success, and predict patient prognosis. The most common technique used for the detection of disease-associated biomarkers is the enzyme linked immunosorbent assay (ELISA. In an ELISA, primary antibodies are incubated with biological samples containing the biomarker of interest. Then, detectible secondary antibodies conjugated with horseradish peroxidase (HRP bind the primary antibodies. Upon addition of a color-changing substrate, the samples provide a colorimetric signal that directly correlates to the targeted biomarker concentration. While ELISAs are effective for analyzing samples with high biomarker content, they lack the sensitivity required to analyze samples with low antigen levels. We hypothesized that the sensitivity of ELISAs could be enhanced by replacing freely delivered primary antibodies with antibody-nanoparticle conjugates that provide excess binding sites for detectible secondary antibodies, ultimately leading to increased signal. Here, we investigated the use of nanoshells (NS decorated with antibodies specific to epidermal growth factor receptor (EGFR as a model system (EGFR-NS. We incubated one healthy and two breast cancer cell lines, each expressing different levels of EGFR, with EGFR-NS, untargeted NS, or unconjugated EGFR antibodies, as well as detectable secondary antibodies. We found that EGFR-NS consistently increased signal intensity relative to unconjugated EGFR antibodies, with a substantial 13-fold enhancement from cells expressing high levels of EGFR. Additionally, 40x more unconjugated antibodies were required to detect EGFR compared to those conjugated to NS. Our results demonstrate that antibody-nanoparticle conjugates lower the detection limit of traditional ELISAs and support further investigation of this strategy with other antibodies and nanoparticles. Owing to their enhanced sensitivity, we anticipate that

  18. Defining New Therapeutics Using a More Immunocompetent Mouse Model of Antibody-Enhanced Dengue Virus Infection

    Science.gov (United States)

    Pinto, Amelia K.; Brien, James D.; Lam, Chia-Ying Kao; Johnson, Syd; Chiang, Cindy; Hiscott, John; Sarathy, Vanessa V.; Barrett, Alan D.; Shresta, Sujan

    2015-01-01

    ABSTRACT With over 3.5 billion people at risk and approximately 390 million human infections per year, dengue virus (DENV) disease strains health care resources worldwide. Previously, we and others established models for DENV pathogenesis in mice that completely lack subunits of the receptors (Ifnar and Ifngr) for type I and type II interferon (IFN) signaling; however, the utility of these models is limited by the pleotropic effect of these cytokines on innate and adaptive immune system development and function. Here, we demonstrate that the specific deletion of Ifnar expression on subsets of murine myeloid cells (LysM Cre+ Ifnarflox/flox [denoted as Ifnarf/f herein]) resulted in enhanced DENV replication in vivo. The administration of subneutralizing amounts of cross-reactive anti-DENV monoclonal antibodies to LysM Cre+ Ifnarf/f mice prior to infection with DENV serotype 2 or 3 resulted in antibody-dependent enhancement (ADE) of infection with many of the characteristics associated with severe DENV disease in humans, including plasma leakage, hypercytokinemia, liver injury, hemoconcentration, and thrombocytopenia. Notably, the pathogenesis of severe DENV-2 or DENV-3 infection in LysM Cre+ Ifnarf/f mice was blocked by pre- or postexposure administration of a bispecific dual-affinity retargeting molecule (DART) or an optimized RIG-I receptor agonist that stimulates innate immune responses. Our findings establish a more immunocompetent animal model of ADE of infection with multiple DENV serotypes in which disease is inhibited by treatment with broad-spectrum antibody derivatives or innate immune stimulatory agents. PMID:26374123

  19. Antibody-dependent NK cell activation is associated with late kidney allograft dysfunction and the complement-independent alloreactive potential of donor-specific antibodies

    Directory of Open Access Journals (Sweden)

    Tristan Legris

    2016-08-01

    Full Text Available Although kidney transplantation remains the best treatment for end-stage renal failure, it is limited by chronic humoral aggression of the graft vasculature by donor-specific antibodies (DSAs. The complement-independent mechanisms that lead to the antibody-mediated rejection (ABMR of kidney allografts remain poorly understood. Increasing lines of evidence have revealed the relevance of natural killer (NK cells as innate immune effectors of antibody-dependent cellular cytotoxicity, but few studies have investigated their alloreactive potential in the context of solid organ transplantation. Our study aimed to investigate the potential contribution of the antibody-dependent alloreactive function of NK cells to kidney graft dysfunction. We first conducted an observational study to investigate whether the cytotoxic function of NK cells is associated with chronic allograft dysfunction. The NK-Cellular Humoral Activation Test (NK-CHAT was designed to evaluate the recipient and antibody-dependent reactivity of NK cells against allogeneic target cells. The release of CD107a/Lamp1+ cytotoxic granules, resulting from the recognition of rituximab-coated B cells by NK cells, was analyzed in 148 kidney transplant recipients (KTRs, mean graft duration: 6.2 years. Enhanced ADCC responsiveness was associated with reduced graft function and identified as an independent risk factor predicting a decline in the estimated glomerular filtration rate (eGFR over a 1-year period (hazard ratio: 2.83. In a second approach, we used the NK-CHAT to reveal the cytotoxic potential of circulating alloantibodies in vitro. The level of CD16 engagement resulting from the in vitro recognition of serum-coated allogeneic B cells or splenic cells was further identified as a specific marker of DSA-induced ADCC. The NK-CHAT scoring of sera obtained from 40 patients at the time of transplant biopsy was associated with ABMR diagnosis. Our findings indicate that despite the administration

  20. Enhancement of anamnestic immunospecific antibody response in orally immunized chickens

    DEFF Research Database (Denmark)

    Mayo, Susan; Carlsson, Hans-Erik; Zagon, Andrea

    2008-01-01

    Production of immunospecific egg yolk antibodies (IgY antibodies) in egg laying hens through oral immunization is an attractive alternative to conventional antibody production in mammals for economic reasons as well as for animal welfare reasons. Oral immunization results in a systemic humoral...... response, but oral booster immunizations lack efficiency. The aim of the present study was to develop immunization schemes in which the concentration of immunospecific IgY would increase following oral booster immunizations. Two groups of egg laying hens (5 in each group) were immunized orally (each...... and one oral dose with BSA+RV. The eggs of the chickens in this group had a significantly higher immunospecific anti BSA IgY-concentration than did any of the eggs from the orally immunized chickens. One of the immunization regimes (immunizations in weeks 1, 7 and 18) clearly included a booster effect...

  1. An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function of a Human Antibody

    Energy Technology Data Exchange (ETDEWEB)

    Krause, Jens C.; Ekiert, Damian C.; Tumpey, Terrence M.; Smith, Patricia B.; Wilson, Ian A.; Crowe, Jr., James E. (Vanderbilt); (Scripps); (CDC)

    2011-09-02

    The structural and functional significance of somatic insertions and deletions in antibody chains is unclear. Here, we demonstrate that a naturally occurring three-amino-acid insertion within the influenza virus-specific human monoclonal antibody 2D1 heavy-chain variable region reconfigures the antibody-combining site and contributes to its high potency against the 1918 and 2009 pandemic H1N1 influenza viruses. The insertion arose through a series of events, including a somatic point mutation in a predicted hot-spot motif, introduction of a new hot-spot motif, a molecular duplication due to polymerase slippage, a deletion due to misalignment, and additional somatic point mutations. Atomic resolution structures of the wild-type antibody and a variant in which the insertion was removed revealed that the three-amino-acid insertion near the base of heavy-chain complementarity-determining region (CDR) H2 resulted in a bulge in that loop. This enlarged CDR H2 loop impinges on adjacent regions, causing distortion of the CDR H1 architecture and its displacement away from the antigen-combining site. Removal of the insertion restores the canonical structure of CDR H1 and CDR H2, but binding, neutralization activity, and in vivo activity were reduced markedly because of steric conflict of CDR H1 with the hemagglutinin antigen.

  2. Enhancement of Monoclonal Antibody Production by Lysine-Containing Peptides

    Czech Academy of Sciences Publication Activity Database

    Franěk, František; Eckschlager, T.; Hermann, K.

    2003-01-01

    Roč. 19, č. 1 (2003), s. 169-174 ISSN 8756-7938 R&D Projects: GA MŠk OC 844.10 Institutional research plan: CEZ:AV0Z5038910; CEZ:MSM 111300005 Keywords : Monoclonal Antibody * Lysine-Containing Peptides Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.488, year: 2003

  3. Antiglycopeptide Mouse Monoclonal Antibody LpMab-21 Exerts Antitumor Activity Against Human Podoplanin Through Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity.

    Science.gov (United States)

    Kato, Yukinari; Kunita, Akiko; Fukayama, Masashi; Abe, Shinji; Nishioka, Yasuhiko; Uchida, Hiroaki; Tahara, Hideaki; Yamada, Shinji; Yanaka, Miyuki; Nakamura, Takuro; Saidoh, Noriko; Yoshida, Kanae; Fujii, Yuki; Honma, Ryusuke; Takagi, Michiaki; Ogasawara, Satoshi; Murata, Takeshi; Kaneko, Mika K

    2017-02-01

    The interaction between podoplanin (PDPN) and C-type lectin-like receptor 2 (CLEC-2) is involved in tumor malignancy. We have established many monoclonal antibodies (mAbs) against human podoplanin using the cancer-specific mAb (CasMab) technology. LpMab-21, one of the mouse antipodoplanin mAbs, is of the IgG 2a subclass, and its minimum epitope was determined to be Thr76-Arg79 of the human podoplanin. Importantly, sialic acid is linked to Thr76; therefore, LpMab-21 is an antiglycopeptide mAb (GpMab). In this study, we investigated whether LpMab-21 shows antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cell lines in vitro and also studied its antitumor activities using a xenograft model. LpMab-21 showed high ADCC and CDC activities against not only podoplanin-expressing Chinese hamster ovary cells but also LN319 glioblastoma cells and PC-10 lung cancer cells, both of which endogenously express podoplanin. Furthermore, LpMab-21 decreased tumor growth in vivo, indicating that LpMab-21 could be useful for antibody therapy against human podoplanin-expressing cancers.

  4. Age Dependence and Isotype Specificity of Influenza Virus Hemagglutinin Stalk-Reactive Antibodies in Humans

    Directory of Open Access Journals (Sweden)

    Raffael Nachbagauer

    2016-01-01

    Full Text Available Influenza remains a major global health burden. Seasonal vaccines offer protection but can be rendered less effective when the virus undergoes extensive antigenic drift. Antibodies that target the highly conserved hemagglutinin stalk can protect against drifted viruses, and vaccine constructs designed to induce such antibodies form the basis for a universal influenza virus vaccine approach. In this study, we analyzed baseline and postvaccination serum samples of children (6 to 59 months, adults (18 to 49 years, and elderly individuals (≥65 years who participated in clinical trials with a recombinant hemagglutinin-based vaccine. We found that baseline IgG and IgA antibodies against the H1 stalk domain correlated with the ages of patients. Children generally had very low baseline titers and did not respond well to the vaccine in terms of making stalk-specific antibodies. Adults showed the highest induction of stalk-specific antibodies, but the elderly had the highest absolute antibody titers against the stalk. Importantly, the stalk antibodies measured by enzyme-linked immunosorbent assay (ELISA showed neutralizing activity in neutralization assays and protected mice in a passive-transfer model in a stalk titer-dependent manner. Finally, we found similar patterns of stalk-specific antibodies directed against the H3 and influenza B virus hemagglutinins, albeit at lower levels than those measured against the H1 stalk. The relatively high levels of stalk-specific antibodies in the elderly patients may explain the previously reported low influenza virus infection rates in this age group. (This study has been registered at ClinicalTrials.gov under registration no. NCT00336453, NCT00539981, and NCT00395174.

  5. High-content Analysis of Antibody Phage-display Library Selection Outputs Identifies Tumor Selective Macropinocytosis-dependent Rapidly Internalizing Antibodies*

    Science.gov (United States)

    Ha, Kevin D.; Bidlingmaier, Scott M.; Zhang, Yafeng; Su, Yang; Liu, Bin

    2014-01-01

    Many forms of antibody-based targeted therapeutics, including antibody drug conjugates, utilize the internalizing function of the targeting antibody to gain intracellular entry into tumor cells. Ideal antibodies for developing such therapeutics should be capable of both tumor-selective binding and efficient endocytosis. The macropinocytosis pathway is capable of both rapid and bulk endocytosis, and recent studies have demonstrated that it is selectively up-regulated by cancer cells. We hypothesize that receptor-dependent macropinocytosis can be achieved using tumor-targeting antibodies that internalize via the macropinocytosis pathway, improving potency and selectivity of the antibody-based targeted therapeutic. Although phage antibody display libraries have been utilized to find antibodies that bind and internalize to target cells, no methods have been described to screen for antibodies that internalize specifically via macropinocytosis. We hereby describe a novel screening strategy to identify phage antibodies that bind and rapidly enter tumor cells via macropinocytosis. We utilized an automated microscopic imaging-based, High Content Analysis platform to identify novel internalizing phage antibodies that colocalize with macropinocytic markers from antibody libraries that we have generated previously by laser capture microdissection-based selection, which are enriched for internalizing antibodies binding to tumor cells in situ residing in their tissue microenvironment (Ruan, W., Sassoon, A., An, F., Simko, J. P., and Liu, B. (2006) Identification of clinically significant tumor antigens by selecting phage antibody library on tumor cells in situ using laser capture microdissection. Mol. Cell. Proteomics. 5, 2364–2373). Full-length human IgG molecules derived from macropinocytosing phage antibodies retained the ability to internalize via macropinocytosis, validating our screening strategy. The target antigen for a cross-species binding antibody with a highly

  6. High-content analysis of antibody phage-display library selection outputs identifies tumor selective macropinocytosis-dependent rapidly internalizing antibodies.

    Science.gov (United States)

    Ha, Kevin D; Bidlingmaier, Scott M; Zhang, Yafeng; Su, Yang; Liu, Bin

    2014-12-01

    Many forms of antibody-based targeted therapeutics, including antibody drug conjugates, utilize the internalizing function of the targeting antibody to gain intracellular entry into tumor cells. Ideal antibodies for developing such therapeutics should be capable of both tumor-selective binding and efficient endocytosis. The macropinocytosis pathway is capable of both rapid and bulk endocytosis, and recent studies have demonstrated that it is selectively up-regulated by cancer cells. We hypothesize that receptor-dependent macropinocytosis can be achieved using tumor-targeting antibodies that internalize via the macropinocytosis pathway, improving potency and selectivity of the antibody-based targeted therapeutic. Although phage antibody display libraries have been utilized to find antibodies that bind and internalize to target cells, no methods have been described to screen for antibodies that internalize specifically via macropinocytosis. We hereby describe a novel screening strategy to identify phage antibodies that bind and rapidly enter tumor cells via macropinocytosis. We utilized an automated microscopic imaging-based, High Content Analysis platform to identify novel internalizing phage antibodies that colocalize with macropinocytic markers from antibody libraries that we have generated previously by laser capture microdissection-based selection, which are enriched for internalizing antibodies binding to tumor cells in situ residing in their tissue microenvironment (Ruan, W., Sassoon, A., An, F., Simko, J. P., and Liu, B. (2006) Identification of clinically significant tumor antigens by selecting phage antibody library on tumor cells in situ using laser capture microdissection. Mol. Cell. Proteomics. 5, 2364-2373). Full-length human IgG molecules derived from macropinocytosing phage antibodies retained the ability to internalize via macropinocytosis, validating our screening strategy. The target antigen for a cross-species binding antibody with a highly active

  7. Defining New Therapeutics Using a More Immunocompetent Mouse Model of Antibody-Enhanced Dengue Virus Infection.

    Science.gov (United States)

    Pinto, Amelia K; Brien, James D; Lam, Chia-Ying Kao; Johnson, Syd; Chiang, Cindy; Hiscott, John; Sarathy, Vanessa V; Barrett, Alan D; Shresta, Sujan; Diamond, Michael S

    2015-09-15

    With over 3.5 billion people at risk and approximately 390 million human infections per year, dengue virus (DENV) disease strains health care resources worldwide. Previously, we and others established models for DENV pathogenesis in mice that completely lack subunits of the receptors (Ifnar and Ifngr) for type I and type II interferon (IFN) signaling; however, the utility of these models is limited by the pleotropic effect of these cytokines on innate and adaptive immune system development and function. Here, we demonstrate that the specific deletion of Ifnar expression on subsets of murine myeloid cells (LysM Cre(+) Ifnar(flox/flox) [denoted as Ifnar(f/f) herein]) resulted in enhanced DENV replication in vivo. The administration of subneutralizing amounts of cross-reactive anti-DENV monoclonal antibodies to LysM Cre(+) Ifnar(f/f) mice prior to infection with DENV serotype 2 or 3 resulted in antibody-dependent enhancement (ADE) of infection with many of the characteristics associated with severe DENV disease in humans, including plasma leakage, hypercytokinemia, liver injury, hemoconcentration, and thrombocytopenia. Notably, the pathogenesis of severe DENV-2 or DENV-3 infection in LysM Cre(+) Ifnar(f/f) mice was blocked by pre- or postexposure administration of a bispecific dual-affinity retargeting molecule (DART) or an optimized RIG-I receptor agonist that stimulates innate immune responses. Our findings establish a more immunocompetent animal model of ADE of infection with multiple DENV serotypes in which disease is inhibited by treatment with broad-spectrum antibody derivatives or innate immune stimulatory agents. Although dengue virus (DENV) infects hundreds of millions of people annually and results in morbidity and mortality on a global scale, there are no approved antiviral treatments or vaccines. Part of the difficulty in evaluating therapeutic candidates is the lack of small animal models that are permissive to DENV and recapitulate the clinical features

  8. Anti-LFA-1 antibodies enhance metastasis of ocular lymphoma to the brain and contralateral eye.

    Science.gov (United States)

    Hochman, Jacob; Shen, DeFen; Gottesman, Michael M; Chan, Chi-Chao

    2013-01-01

    Previously we demonstrated that intraperitoneal (IP) inoculation of Rev-2-T-6 mouse lymphoma into syngeneic Balb/c hosts resulted in brain metastasis, migration along the optic nerve sheath, and ocular infiltration. In a second model: intravitreal inoculation of Rev-2-T-6 cells, the developing lymphoma was largely confined within the eye, seldom breaching the retinal pigment epithelium to reside in the choroid and sclera. There was no retrograde infiltration into the brain. Here, we describe a third, complementary model, whereby intravitreal inoculation of Rev-2-T-6 cells into Balb/c mice, followed by repeated IP inoculations of anti-LFA-1/CD11a monoclonal antibodies, results in extensive infiltration of the choroid, sclera, conjunctiva, eyelids and orbit. Furthermore, the lymphoma cells metastasize along the optic nerve sheath into the brain, and through the contralateral optic nerve tract into the contralateral eye. There is no systemic involvement of the lymphoma. Furthermore, anti-LFA-1 treatment results in elevated levels of serum anti-Rev-2-T-6 antibodies. Inoculation of Rev-2-T-6 cells into the vitreous of severe combined immune deficient mice demonstrates a course of clinical signs and histopathological findings similar to those in immune-competent mice treated with anti-LFA-1 antibodies, including invasion of the contralateral eye. Taken together, these findings suggest that confinement of Rev-2-T-6 lymphoma cells to the eye depends on active immune surveillance using a population of effector cells expressing the cell surface integrin LFA-1. Impairing this protection enhances tumor aggressiveness within the eye, and the likelihood of early retrograde lymphoma metastasis into the brain and the contralateral eye.

  9. Enhancing Stability of Camelid and Shark Single Domain Antibodies: An Overview

    Directory of Open Access Journals (Sweden)

    Ellen R. Goldman

    2017-07-01

    Full Text Available Single domain antibodies (sdAbs are gaining a reputation as superior recognition elements as they combine the advantages of the specificity and affinity found in conventional antibodies with high stability and solubility. Melting temperatures (Tms of sdAbs cover a wide range from below 50 to over 80°C. Many sdAbs have been engineered to increase their Tm, making them stable until exposed to extreme temperatures. SdAbs derived from the variable heavy chains of camelid and shark heavy chain-only antibodies are termed VHH and VNAR, respectively, and generally exhibit some ability to refold and bind antigen after heat denaturation. This ability to refold varies from 0 to 100% and is a property dependent on both intrinsic factors of the sdAb and extrinsic conditions such as the sample buffer ionic strength, pH, and sdAb concentration. SdAbs have also been engineered to increase their solubility and refolding ability, which enable them to function even after exposure to temperatures that exceed their melting point. In addition, efforts to improve their stability at extreme pH and in the presence of chemical denaturants or proteases have been undertaken. Multiple routes have been employed to engineer sdAbs with these enhanced stabilities. The methods utilized to achieve these goals include grafting complementarity-determining regions onto stable frameworks, introduction of non-canonical disulfide bonds, random mutagenesis combined with stringent selection, point mutations such as inclusion of negative charges, and genetic fusions. Increases of up to 20°C have been realized, pushing the Tm of some sdAbs to over 90°C. Herein, we present an overview of the work done to stabilize sdAbs derived from camelids and sharks. Utilizing these various strategies sdAbs have been stabilized without significantly compromising their affinity, thereby providing superior reagents for detection, diagnostic, and therapeutic applications.

  10. Age Dependence and Isotype Specificity of Influenza Virus Hemagglutinin Stalk-Reactive Antibodies in Humans.

    Science.gov (United States)

    Nachbagauer, Raffael; Choi, Angela; Izikson, Ruvim; Cox, Manon M; Palese, Peter; Krammer, Florian

    2016-01-19

    Influenza remains a major global health burden. Seasonal vaccines offer protection but can be rendered less effective when the virus undergoes extensive antigenic drift. Antibodies that target the highly conserved hemagglutinin stalk can protect against drifted viruses, and vaccine constructs designed to induce such antibodies form the basis for a universal influenza virus vaccine approach. In this study, we analyzed baseline and postvaccination serum samples of children (6 to 59 months), adults (18 to 49 years), and elderly individuals (≥65 years) who participated in clinical trials with a recombinant hemagglutinin-based vaccine. We found that baseline IgG and IgA antibodies against the H1 stalk domain correlated with the ages of patients. Children generally had very low baseline titers and did not respond well to the vaccine in terms of making stalk-specific antibodies. Adults showed the highest induction of stalk-specific antibodies, but the elderly had the highest absolute antibody titers against the stalk. Importantly, the stalk antibodies measured by enzyme-linked immunosorbent assay (ELISA) showed neutralizing activity in neutralization assays and protected mice in a passive-transfer model in a stalk titer-dependent manner. Finally, we found similar patterns of stalk-specific antibodies directed against the H3 and influenza B virus hemagglutinins, albeit at lower levels than those measured against the H1 stalk. The relatively high levels of stalk-specific antibodies in the elderly patients may explain the previously reported low influenza virus infection rates in this age group. (This study has been registered at ClinicalTrials.gov under registration no. NCT00336453, NCT00539981, and NCT00395174.) The present study provides evidence that titers of broadly neutralizing hemagglutinin stalk-reactive antibodies increase with age, possibly due to repeated exposure to divergent influenza viruses. These relatively high levels of antistalk titers may be

  11. Immunization with Clinical HIV-1 Env Proteins Induces Broad Antibody Dependent Cellular Cytotoxicity-Mediating Antibodies in a Rabbit Vaccination Model

    DEFF Research Database (Denmark)

    Karlsson, Ingrid; Borggren, Marie; Jensen, Sanne Skov

    2018-01-01

    The induction of both neutralizing antibodies and non-neutralizing antibodies with effector functions, for example, antibody-dependent cellular cytotoxicity (ADCC), is desired in the search for effective vaccines against HIV-1. In the pursuit of novel immunogens capable of inducing an efficient...... of inducing antibodies with ADCC activity did not necessarily induce neutralizing activity and vice versa. Nevertheless, we identified vaccine candidates that were able to concurrently induce both types of responses and that had ADCC activity that was cross-reactive between different subtypes. When searching...

  12. Luminol-dependent chemiluminescence in antibody-sensitized neutrophils stimulated with protein A-bearing staphylococci.

    Science.gov (United States)

    Nishihara, S; Seki, K; Ikigai, H; Masuda, S

    1988-01-01

    When mouse polymorphonuclear leukocytes (PMNs) sensitized with rabbit antibody to mouse Ehrlich ascites tumor cells were stimulated by Staphylococcus aureus Cowan I cells, a conspicuous luminol-dependent chemiluminescence was observed in the absence of opsonin. The profile of the chemiluminescence (CL) response evoked by staphylococcal cells from antibody-sensitized PMNs had two peaks. An initial peak, observed within 1 min after stimulation, was sharp and high and a second peak, observed about 5 min after stimulation, was low and extended. The CL response of antibody-sensitized PMNs stimulated by S. aureus Cowan I cells was dose-dependently blocked by preincubation with soluble SpA. Cells of a mutant derived from S. aureus Cowan I strain with trace amounts of cell-bound SpA failed to stimulate the antibody-sensitized PMNs to generate the CL response. The antibody-sensitized PMNs were found to phagocytize SpA-bearing S. aureus cells even in the absence of opsonic serum. These results suggest that the observation presented here might provide a useful tool for the investigation of CL response of PMNs.

  13. Enhanced monoclonal antibody production by gradual increase of osmotic pressure

    OpenAIRE

    Lin, Jianqiang; Takagi, Mutsumi; Qu, Yinbo; Gao, Peiji; Yoshida, Toshiomi

    1999-01-01

    The time length required for the adaptation of AFP-27 hybridoma cells to high osmotic pressure and the effect of a gradual increase of osmotic pressure on monoclonal antibody production were investigated. When the cells were subjected to an increase of osmotic pressure from 300 mOsmol kg-1 to 366 mOsmol kg- 1, the intracellular content of osmoprotective free amino acids reached a maximum level 6 h after the osmotic pressure was increased to 366 mOsmol kg-1. The same time period of 6 h incubat...

  14. Enhancing antibody patent protection using epitope mapping information

    Science.gov (United States)

    Deng, Xiaoxiang; Storz, Ulrich; Doranz, Benjamin J.

    2018-01-01

    ABSTRACT As the $100B therapeutic monoclonal antibody (mAb) market continues to grow, developers of therapeutic mAbs increasingly face the need to strengthen patent protection of their products and enforce their patents in courts. In view of changes in the patent law landscape, patent applications are strategically using information on the precise binding sites of their mAbs, i.e., the epitopes, to support patent novelty, non-obviousness, subject matter, and a tightened written description requirement for broad genus antibody claims. Epitope data can also allow freedom-to-operate for second-generation mAbs by differentiation from patented first-generation mAbs. Numerous high profile court cases, including Amgen v. Sanofi over rival mAbs that block PCSK9 activity, have been centered on epitope mapping claims, highlighting the importance of epitopes in determining broad mAb patent rights. Based on these cases, epitope mapping claims must describe a sufficiently large number of mAbs that share an epitope, and each epitope must be described at amino acid resolution. Here, we review current best practices for the use of epitope information to overcome the increasing challenges of patenting mAbs, and how the quality, conformation, and resolution of epitope residue data can influence the breadth and strength of mAb patents. PMID:29120697

  15. Enhancing antibody patent protection using epitope mapping information.

    Science.gov (United States)

    Deng, Xiaoxiang; Storz, Ulrich; Doranz, Benjamin J

    As the $100B therapeutic monoclonal antibody (mAb) market continues to grow, developers of therapeutic mAbs increasingly face the need to strengthen patent protection of their products and enforce their patents in courts. In view of changes in the patent law landscape, patent applications are strategically using information on the precise binding sites of their mAbs, i.e., the epitopes, to support patent novelty, non-obviousness, subject matter, and a tightened written description requirement for broad genus antibody claims. Epitope data can also allow freedom-to-operate for second-generation mAbs by differentiation from patented first-generation mAbs. Numerous high profile court cases, including Amgen v. Sanofi over rival mAbs that block PCSK9 activity, have been centered on epitope mapping claims, highlighting the importance of epitopes in determining broad mAb patent rights. Based on these cases, epitope mapping claims must describe a sufficiently large number of mAbs that share an epitope, and each epitope must be described at amino acid resolution. Here, we review current best practices for the use of epitope information to overcome the increasing challenges of patenting mAbs, and how the quality, conformation, and resolution of epitope residue data can influence the breadth and strength of mAb patents.

  16. A Mouse Monoclonal Antibody against Dengue Virus Type 1 Mochizuki Strain Targeting Envelope Protein Domain II and Displaying Strongly Neutralizing but Not Enhancing Activity

    Science.gov (United States)

    Kotaki, Tomohiro; Konishi, Eiji

    2013-01-01

    Dengue fever and its more severe form, dengue hemorrhagic fever, are major global concerns. Infection-enhancing antibodies are major factors hypothetically contributing to increased disease severity. In this study, we generated 26 monoclonal antibodies (MAbs) against the dengue virus type 1 Mochizuki strain. We selected this strain because a relatively large number of unique and rare amino acids were found on its envelope protein. Although most MAbs showing neutralizing activities exhibited enhancing activities at subneutralizing doses, one MAb (D1-IV-7F4 [7F4]) displayed neutralizing activities without showing enhancing activities at lower concentrations. In contrast, another MAb (D1-V-3H12 [3H12]) exhibited only enhancing activities, which were suppressed by pretreatment of cells with anti-FcγRIIa. Although antibody engineering revealed that antibody subclass significantly affected 7F4 (IgG3) and 3H12 (IgG1) activities, neutralizing/enhancing activities were also dependent on the epitope targeted by the antibody. 7F4 recognized an epitope on the envelope protein containing E118 (domain II) and had a neutralizing activity 10- to 1,000-fold stronger than the neutralizing activity of previously reported human or humanized neutralizing MAbs targeting domain I and/or domain II. An epitope-blocking enzyme-linked immunosorbent assay (ELISA) indicated that a dengue virus-immune population possessed antibodies sharing an epitope with 7F4. Our results demonstrating induction of these antibody species (7F4 and 3H12) in Mochizuki-immunized mice may have implications for dengue vaccine strategies designed to minimize induction of enhancing antibodies in vaccinated humans. PMID:24049185

  17. Beta2-glycoprotein I dependent anticardiolipin antibodies and lupus anticoagulant in patients with recurrent pregnancy loss.

    Directory of Open Access Journals (Sweden)

    Kumar K

    2002-01-01

    Full Text Available AIM: The present study was aimed to define the incidence of antiphospholipid antibodies of different types lupus anticoagulant (LAC, venereal disease research laboratory test (VDRL and Beta2-glycoprotein I dependent anticardiolipin antibodies Beta2 I aCL in our cohort of population experiencing recurrent pregnancy loss (RPL from Andhra Pradesh, South India. SETTING AND DESIGN: A referral case-control study at a tertiary centre over a period of 5 years. PARTICIPANTS: 150 couples experiencing 3 or more recurrent pregnancy losses with similar number of matched controls. MATERIAL AND METHODS: LAC activity was measured by the activated partial thromboplastin time (aPTT according to the method of Proctor and Rapaport with relevant modifications. VDRL analysis was performed by the kit method supplied by Ranbaxy Diagnostics Limited and Beta2 Glycoprotein I dependent anticardiolipin antibodies were estimated by ELISA kit (ORGen Tech, GmbH, Germany with human Beta2 Glycoprotein I as co-factor. STATISTICAL ANALYSIS: Statistical analysis was performed using Student′s t test. RESULTS: LAC activity was found positive in 11 women (10.28%. The mean +/- SE Beta2 I aCL concentration in the study group was 14.53 (micro/ml +/- 1.79 (range 0 to 90.4 micro/ml which was higher than the control group with a mean +/- SE of 7.26 (micro/ml +/- 0.40 (range 0 to 18 u/ml. The binding of the antibodies to the antigen was observed in 40.24% (n=33 of the cases compared to 6.09% (n=5 in controls. VDRL test was positive in 7(2.34% individuals (3 couples and 1 male partner and none among controls. CONCLUSIONS: The present study indicates the importance of antiphospholipid antibodies in women experiencing RPL and suggests the usefulness of screening for these antibodies as a mandatory routine for instituting efficient therapeutic regimens for a successful outcome of pregnancy.

  18. Adjuvant-Mediated Epitope Specificity and Enhanced Neutralizing Activity of Antibodies Targeting Dengue Virus Envelope Protein

    Directory of Open Access Journals (Sweden)

    Denicar Lina Nascimento Fabris Maeda

    2017-09-01

    Full Text Available The heat-labile toxins (LT produced by enterotoxigenic Escherichia coli display adjuvant effects to coadministered antigens, leading to enhanced production of serum antibodies. Despite extensive knowledge of the adjuvant properties of LT derivatives, including in vitro-generated non-toxic mutant forms, little is known about the capacity of these adjuvants to modulate the epitope specificity of antibodies directed against antigens. This study characterizes the role of LT and its non-toxic B subunit (LTB in the modulation of antibody responses to a coadministered antigen, the dengue virus (DENV envelope glycoprotein domain III (EDIII, which binds to surface receptors and mediates virus entry into host cells. In contrast to non-adjuvanted or alum-adjuvanted formulations, antibodies induced in mice immunized with LT or LTB showed enhanced virus-neutralization effects that were not ascribed to a subclass shift or antigen affinity. Nonetheless, immunosignature analyses revealed that purified LT-adjuvanted EDIII-specific antibodies display distinct epitope-binding patterns with regard to antibodies raised in mice immunized with EDIII or the alum-adjuvanted vaccine. Notably, the analyses led to the identification of a specific EDIII epitope located in the EF to FG loop, which is involved in the entry of DENV into eukaryotic cells. The present results demonstrate that LT and LTB modulate the epitope specificity of antibodies generated after immunization with coadministered antigens that, in the case of EDIII, was associated with the induction of neutralizing antibody responses. These results open perspectives for the more rational development of vaccines with enhanced protective effects against DENV infections.

  19. Subversion of innate defenses by the interplay between DENV and pre-existing enhancing antibodies: TLRs signaling collapse.

    Science.gov (United States)

    Modhiran, Naphak; Kalayanarooj, Siripen; Ubol, Sukathida

    2010-12-21

    The phenomenon of antibody dependent enhancement as a major determinant that exacerbates disease severity in DENV infections is well accepted. While the detailed mechanism of antibody enhanced disease severity is unclear, evidence suggests that it is associated with both increased DENV infectivity and suppression of the type I IFN and pro-inflammatory cytokine responses. Therefore, it is imperative for us to understand the intracellular mechanisms altered during ADE infection to decipher the mechanism of severe pathogenesis. In this present work, qRT-PCR, immunoblotting and gene array analysis were conducted to determine whether DENV-antibody complex infection exerts a suppressive effect on the expression and/or function of the pathogen recognition patterns, focusing on the TLR-signaling pathway. We show here that FcγRI and FcγRIIa synergistically facilitated entry of DENV-antibody complexes into monocytic THP-1 cells. Ligation between DENV-antibody complexes and FcR not only down regulated TLRs gene expression but also up regulated SARM, TANK, and negative regulators of the NF-κB pathway, resulting in suppression of innate responses but increased viral production. These results were confirmed by blocking with anti-FcγRI or anti-FcγRIIa antibodies which reduced viral production, up-regulated IFN-β synthesis, and increased gene expression in the TLR-dependent signaling pathway. The negative impact of DENV-ADE infection on the TLR-dependent pathway was strongly supported by gene array screening which revealed that both MyD88-dependent and -independent signaling molecules were down regulated during DENV-ADE infection. Importantly, the same phenomenon was seen in PBMC of secondary DHF/DSS patients but not in PBMC of DF patients. Our present work demonstrates the mechanism by which DENV uses pre-existing immune mediators to defeat the principal activating pathway of innate defense resulting in suppression of an array of innate immune responses. Interestingly, this

  20. A potent human neutralizing antibody Fc-dependently reduces established HBV infections.

    Science.gov (United States)

    Li, Dan; He, Wenhui; Liu, Ximing; Zheng, Sanduo; Qi, Yonghe; Li, Huiyu; Mao, Fengfeng; Liu, Juan; Sun, Yinyan; Pan, Lijing; Du, Kaixin; Ye, Keqiong; Li, Wenhui; Sui, Jianhua

    2017-09-26

    Hepatitis B virus (HBV) infection is a major global health problem. Currently-available therapies are ineffective in curing chronic HBV infection. HBV and its satellite hepatitis D virus (HDV) infect hepatocytes via binding of the preS1 domain of its large envelope protein to sodium taurocholate cotransporting polypeptide (NTCP). Here, we developed novel human monoclonal antibodies that block the engagement of preS1 with NTCP and neutralize HBV and HDV with high potency. One antibody, 2H5-A14, functions at picomolar level and exhibited neutralization-activity-mediated prophylactic effects. It also acts therapeutically by eliciting antibody-Fc-dependent immunological effector functions that impose durable suppression of viral infection in HBV-infected mice, resulting in reductions in the levels of the small envelope antigen and viral DNA, with no emergence of escape mutants. Our results illustrate a novel antibody-Fc-dependent approach for HBV treatment and suggest 2H5-A14 as a novel clinical candidate for HBV prevention and treatment of chronic HBV infection.

  1. Enhanced inflammation in New Zealand white rabbits when MERS-CoV reinfection occurs in the absence of neutralizing antibody.

    Directory of Open Access Journals (Sweden)

    Katherine V Houser

    2017-08-01

    Full Text Available The Middle East respiratory syndrome coronavirus (MERS-CoV is a zoonotic betacoronavirus that was first detected in humans in 2012 as a cause of severe acute respiratory disease. As of July 28, 2017, there have been 2,040 confirmed cases with 712 reported deaths. While many infections have been fatal, there have also been a large number of mild or asymptomatic cases discovered through monitoring and contact tracing. New Zealand white rabbits are a possible model for asymptomatic infection with MERS-CoV. In order to discover more about non-lethal infections and to learn whether a single infection with MERS-CoV would protect against reinfection, we inoculated rabbits with MERS-CoV and monitored the antibody and inflammatory response. Following intranasal infection, rabbits developed a transient dose-dependent pulmonary infection with moderately high levels of viral RNA, viral antigen, and perivascular inflammation in multiple lung lobes that was not associated with clinical signs. The rabbits developed antibodies against viral proteins that lacked neutralizing activity and the animals were not protected from reinfection. In fact, reinfection resulted in enhanced pulmonary inflammation, without an associated increase in viral RNA titers. Interestingly, passive transfer of serum from previously infected rabbits to naïve rabbits was associated with enhanced inflammation upon infection. We further found this inflammation was accompanied by increased recruitment of complement proteins compared to primary infection. However, reinfection elicited neutralizing antibodies that protected rabbits from subsequent viral challenge. Our data from the rabbit model suggests that people exposed to MERS-CoV who fail to develop a neutralizing antibody response, or persons whose neutralizing antibody titers have waned, may be at risk for severe lung disease on re-exposure to MERS-CoV.

  2. HIV-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC) -Mediating Antibodies Decline while NK Cell Function Increases during Antiretroviral Therapy (ART)

    DEFF Research Database (Denmark)

    Skov Jensen, Sanne; Fomsgaard, Anders; Borggren, Marie

    2015-01-01

    Understanding alterations in HIV-specific immune responses during antiretroviral therapy (ART), such as antibody-dependent cellular cytotoxicity (ADCC), is important in the development of novel strategies to control HIV-1 infection. This study included 53 HIV-1 positive individuals. We evaluated...... during ART. These findings suggest improved cytotoxic function of the NK cells if initiating ART early during infection, while the levels of ADCC mediating antibodies declined during ART....

  3. Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Weizao, E-mail: chenw3@mail.nih.gov [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Feng, Yang [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Wang, Yanping [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); The Basic Research Program, Science Applications International Corporation-Frederick, Inc., National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Zhu, Zhongyu; Dimitrov, Dimiter S. [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Some recombinant HIV-1 gp120s do not preserve their conformations on gp140s. Black-Right-Pointing-Pointer We hypothesize that CD4i antibodies could induce conformational changes in gp120. Black-Right-Pointing-Pointer CD4i antibodies enhance binding of CD4 and CD4bs antibodies to gp120. Black-Right-Pointing-Pointer CD4i antibody-gp120 fusion proteins could have potential as vaccine immunogens. -- Abstract: Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.

  4. Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

    International Nuclear Information System (INIS)

    Chen, Weizao; Feng, Yang; Wang, Yanping; Zhu, Zhongyu; Dimitrov, Dimiter S.

    2012-01-01

    Highlights: ► Some recombinant HIV-1 gp120s do not preserve their conformations on gp140s. ► We hypothesize that CD4i antibodies could induce conformational changes in gp120. ► CD4i antibodies enhance binding of CD4 and CD4bs antibodies to gp120. ► CD4i antibody-gp120 fusion proteins could have potential as vaccine immunogens. -- Abstract: Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.

  5. The bacterial enhancer-dependent RNA polymerase.

    Science.gov (United States)

    Zhang, Nan; Darbari, Vidya C; Glyde, Robert; Zhang, Xiaodong; Buck, Martin

    2016-11-01

    Transcription initiation is highly regulated in bacterial cells, allowing adaptive gene regulation in response to environment cues. One class of promoter specificity factor called sigma54 enables such adaptive gene expression through its ability to lock the RNA polymerase down into a state unable to melt out promoter DNA for transcription initiation. Promoter DNA opening then occurs through the action of specialized transcription control proteins called bacterial enhancer-binding proteins (bEBPs) that remodel the sigma54 factor within the closed promoter complexes. The remodelling of sigma54 occurs through an ATP-binding and hydrolysis reaction carried out by the bEBPs. The regulation of bEBP self-assembly into typically homomeric hexamers allows regulated gene expression since the self-assembly is required for bEBP ATPase activity and its direct engagement with the sigma54 factor during the remodelling reaction. Crystallographic studies have now established that in the closed promoter complex, the sigma54 factor occupies the bacterial RNA polymerase in ways that will physically impede promoter DNA opening and the loading of melted out promoter DNA into the DNA-binding clefts of the RNA polymerase. Large-scale structural re-organizations of sigma54 require contact of the bEBP with an amino-terminal glutamine and leucine-rich sequence of sigma54, and lead to domain movements within the core RNA polymerase necessary for making open promoter complexes and synthesizing the nascent RNA transcript. © 2016 The Author(s).

  6. Synthetic Three-Component HIV-1 V3 Glycopeptide Immunogens Induce Glycan-Dependent Antibody Responses.

    Science.gov (United States)

    Cai, Hui; Orwenyo, Jared; Giddens, John P; Yang, Qiang; Zhang, Roushu; LaBranche, Celia C; Montefiori, David C; Wang, Lai-Xi

    2017-12-21

    Eliciting broadly neutralizing antibody (bNAb) responses against HIV-1 is a major goal for a prophylactic HIV-1 vaccine. One approach is to design immunogens based on known broadly neutralizing epitopes. Here we report the design and synthesis of an HIV-1 glycopeptide immunogen derived from the V3 domain. We performed glycopeptide epitope mapping to determine the minimal glycopeptide sequence as the epitope of V3-glycan-specific bNAbs PGT128 and 10-1074. We further constructed a self-adjuvant three-component immunogen that consists of a 33-mer V3 glycopeptide epitope, a universal T helper epitope P30, and a lipopeptide (Pam 3 CSK 4 ) that serves as a ligand of Toll-like receptor 2. Rabbit immunization revealed that the synthetic self-adjuvant glycopeptide could elicit substantial glycan-dependent antibodies that exhibited broader recognition of HIV-1 gp120s than the non-glycosylated V3 peptide. These results suggest that the self-adjuvant synthetic glycopeptides can serve as an important component to elicit glycan-specific antibodies in HIV vaccine design. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Opposite effects of total lymphoid irradiation on T cell-dependent and T cell-independent antibody responses

    Energy Technology Data Exchange (ETDEWEB)

    Tanay, A.; Strober, S.

    1984-02-01

    The effect of total lymphoid irradiation (TLI) on the primary antibody response to the dinitrophenylated heterologous protein, keyhole limpet hemocyanin (DNP-KLH), in complete Freund's adjuvant (CFA), and to the trinitrophenylated polysaccharide antigen, Brucella abortus (TNP-BA), was studied in BALB/c mice. The antibody response to both antigens was diminished in comparison with nonirradiated mice when antigens were injected within 3 days after TLI. When the mice were immunized 30 days after completion of TLI the antibody response to DNP-KLH in CFA was still diminished, but the antibody response to TNP-BA was enhanced 5- to 10-fold as compared with that of control animals. The opposite effect of TLI on the two antibody responses was also observed in a syngeneic primary adoptive transfer system.

  8. Therapeutic efficacy of antibodies lacking Fcγ receptor binding against lethal dengue virus infection is due to neutralizing potency and blocking of enhancing antibodies [corrected].

    Directory of Open Access Journals (Sweden)

    Katherine L Williams

    2013-02-01

    Full Text Available Dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS are life-threatening complications following infection with one of the four serotypes of dengue virus (DENV. At present, no vaccine or antiviral therapies are available against dengue. Here, we characterized a panel of eight human or mouse-human chimeric monoclonal antibodies (MAbs and their modified variants lacking effector function and dissected the mechanism by which some protect against antibody-enhanced lethal DENV infection. We found that neutralizing modified MAbs that recognize the fusion loop or the A strand epitopes on domains II and III of the envelope protein, respectively, act therapeutically by competing with and/or displacing enhancing antibodies. By analyzing these relationships, we developed a novel in vitro suppression-of-enhancement assay that predicts the ability of modified MAbs to act therapeutically against antibody-enhanced disease in vivo. These studies provide new insight into the biology of DENV pathogenesis and the requirements for antibodies to treat lethal DENV disease.

  9. Ca2+-dependent mobility of vesicles capturing anti-VGLUT1 antibodies

    International Nuclear Information System (INIS)

    Stenovec, Matjaz; Kreft, Marko; Grilc, Sonja; Potokar, Maja; Kreft, Mateja Erdani; Pangrsic, Tina; Zorec, Robert

    2007-01-01

    Several aspects of secretory vesicle cycle have been studied in the past, but vesicle trafficking in relation to the fusion site is less well understood. In particular, the mobility of recaptured vesicles that traffic back toward the central cytoplasm is still poorly defined. We exposed astrocytes to antibodies against the vesicular glutamate transporter 1 (VGLUT1), a marker of glutamatergic vesicles, to fluorescently label vesicles undergoing Ca 2+ -dependent exocytosis and examined their number, fluorescence intensity, and mobility by confocal microscopy. In nonstimulated cells, immunolabeling revealed discrete fluorescent puncta, indicating that VGLUT1 vesicles, which are approximately 50 nm in diameter, cycle slowly between the plasma membrane and the cytoplasm. When the cytosolic Ca 2+ level was raised with ionomycin, the number and fluorescence intensity of the puncta increased, likely because the VGLUT1 epitopes were more accessible to the extracellularly applied antibodies following Ca 2+ -triggered exocytosis. In nonstimulated cells, the mobility of labeled vesicles was limited. In stimulated cells, many vesicles exhibited directional mobility that was abolished by cytoskeleton-disrupting agents, indicating dependence on intact cytoskeleton. Our findings show that postfusion vesicle mobility is regulated and may likely play a role in synaptic vesicle cycle, and also more generally in the genesis and removal of endocytic vesicles

  10. Intratumoral delivery of CpG-conjugated anti-MUC1 antibody enhances NK cell anti-tumor activity.

    Science.gov (United States)

    Schettini, Jorge; Kidiyoor, Amritha; Besmer, Dahlia M; Tinder, Teresa L; Roy, Lopamudra Das; Lustgarten, Joseph; Gendler, Sandra J; Mukherjee, Pinku

    2012-11-01

    Monoclonal antibodies (mAbs) against tumor-associated antigens are useful anticancer agents. Antibody-dependent cellular cytotoxicity (ADCC) is one of the major mechanisms responsible for initiating natural killer cell (NK)-mediated killing of tumors. However, the regulation of ADCC via NK cells is poorly understood. We have investigated the cytolytic activity of NK cells against pancreatic cancer cells that were coated with an antibody directed against the human tumor antigen, Mucin-1 designated HMFG-2, either alone or conjugated to CpG oligodeoxynucleotide (CpG ODN). Conjugated antibodies were tested for their ability to elicit ADCC in vitro and in vivo against pancreatic cancer cells. NK cells cultured in the presence of immobilized CpG ODN, HMFG-2 Ab, or CpG ODN-conjugated HMFG-2 Ab were able to up-regulate perforin similarly. Interestingly, a significant higher ADCC was observed when CpG ODN-conjugated HMFG-2-coated tumor cells were co-cultured with NK cells compared to unconjugated HMFG-2 Ab or CpG ODN alone. Moreover, MyD88-deficient NK cells can perform ADCC in vitro. Furthermore, intratumoral injections of CpG ODN-conjugated HMFG-2 induced a significant reduction in tumor burden in vivo in an established model of pancreatic tumor in nude mice compared to CpG ODN or the HMFG-2 alone. Depletion of macrophages or NK cells before treatment confirmed that both cells were required for the anti-tumor response in vivo. Results also suggest that CpG ODN and HMFG-2 Ab could be sensed by NK cells on the mAb-coated tumor cells triggering enhanced ADCC in vitro and in vivo.

  11. Effect of trastuzumab interchain disulfide bond cleavage on Fcγ receptor binding and antibody-dependent tumour cell phagocytosis.

    Science.gov (United States)

    Suzuki, Mami; Yamanoi, Ayaka; Machino, Yusuke; Ootsubo, Michiko; Izawa, Ken-ichi; Kohroki, Junya; Masuho, Yasuhiko

    2016-01-01

    The Fc domain of human IgG1 binds to Fcγ receptors (FcγRs) to induce effector functions such as phagocytosis. There are four interchain disulfide bonds between the H and L chains. In this study, the disulfide bonds within the IgG1 trastuzumab (TRA), which is specific for HER2, were cleaved by mild S-sulfonation or by mild reduction followed by S-alkylation with three different reagents. The cleavage did not change the binding activities of TRA to HER2-bearing SK-BR-3 cells. The binding activities of TRA to FcγRIIA and FcγRIIB were greatly enhanced by modification with mild reduction and S-alkylation with ICH2CONH2 or N-(4-aminophenyl) maleimide, while the binding activities of TRA to FcγRI and FcγRIIIA were decreased by any of the four modifications. However, the interchain disulfide bond cleavage by the different modifications did not change the antibody-dependent cell-mediated phagocytosis (ADCP) of SK-BR-3 cells by activated THP-1 cells. The order of FcγR expression levels on the THP-1 cells was FcγRII > FcγRI > FcγRIII and ADCP was inhibited by blocking antibodies against FcγRI and FcγRII. These results imply that the effect of the interchain disulfide bond cleavage on FcγRs binding and ADCP is dependent on modifications of the cysteine residues and the FcγR isotypes. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  12. Neutralization of antibody-enhanced dengue infection by VIS513, a pan serotype reactive monoclonal antibody targeting domain III of the dengue E protein

    Science.gov (United States)

    Robinson, Luke N.; Ong, Li Ching; Rowley, Kirk J.; Winnett, Alexander; Tan, Hwee Cheng; Hobbie, Sven; Shriver, Zachary; Babcock, Gregory J.; Alonso, Sylvie; Ooi, Eng Eong

    2018-01-01

    Dengue virus (DENV) infection imposes enormous health and economic burden worldwide with no approved treatment. Several small molecules, including lovastatin, celgosivir, balapiravir and chloroquine have been tested for potential anti-dengue activity in clinical trials; none of these have demonstrated a protective effect. Recently, based on identification and characterization of cross-serotype neutralizing antibodies, there is increasing attention on the potential for dengue immunotherapy. Here, we tested the ability of VIS513, an engineered cross-neutralizing humanized antibody targeting the DENV E protein domain III, to overcome antibody-enhanced infection and high but brief viremia, which are commonly encountered in dengue patients, in various in vitro and in vivo models. We observed that VIS513 efficiently neutralizes DENV at clinically relevant viral loads or in the presence of enhancing levels of DENV immune sera. Single therapeutic administration of VIS513 in mouse models of primary infection or lethal secondary antibody-enhanced infection, reduces DENV titers and protects from lethal infection. Finally, VIS513 administration does not readily lead to resistance, either in cell culture systems or in animal models of dengue infection. The findings suggest that rapid viral reduction during acute DENV infection with a monoclonal antibody is feasible. PMID:29425203

  13. Rapidity dependence of strangeness enhancement factor at FAIR energies

    International Nuclear Information System (INIS)

    Dey, Kalyan; Bhattacharjee, B.

    2014-01-01

    Strange particles are produced only at the time of collisions and thus expected to carry important information of collision dynamics. Strangeness enhancement is considered to be one of the traditional signatures of formation of Quark Gluon Plasma (QGP). Due to the limitation of the detector acceptance, the past and ongoing heavy ion experiments could measure the strangeness enhancement at midrapidity only. But the future heavy ion experiment CBM at FAIR will have the access to the entire forward rapidity hemisphere and thus the experimental determination of rapidity dependent strangeness enhancement is a possibility. In this work, an attempt has therefore been made to study the rapidity dependent strangeness enhancement at FAIR energies with the help of a string based hadronic model (UrQMD). A sum of 93 million minimum biased UrQMD events have been used for the present analysis

  14. Heme Oxygenase-1 Inhibits HLA Class I Antibody-Dependent Endothelial Cell Activation.

    Directory of Open Access Journals (Sweden)

    Eva Zilian

    Full Text Available Antibody-mediated rejection (AMR is a key limiting factor for long-term graft survival in solid organ transplantation. Human leukocyte antigen (HLA class I (HLA I antibodies (Abs play a major role in the pathogenesis of AMR via their interactions with HLA molecules on vascular endothelial cells (ECs. The antioxidant enzyme heme oxygenase (HO-1 has anti-inflammatory functions in the endothelium. As complement-independent effects of HLA I Abs can activate ECs, it was the goal of the current study to investigate the role of HO-1 on activation of human ECs by HLA I Abs. In cell cultures of various primary human macro- and microvascular ECs treatment with monoclonal pan- and allele-specific HLA I Abs up-regulated the expression of inducible proinflammatory adhesion molecules and chemokines (vascular cell adhesion molecule-1 [VCAM-1], intercellular cell adhesion molecule-1 [ICAM-1], interleukin-8 [IL-8] and monocyte chemotactic protein 1 [MCP-1]. Pharmacological induction of HO-1 with cobalt-protoporphyrin IX reduced, whereas inhibition of HO-1 with either zinc-protoporphyrin IX or siRNA-mediated knockdown increased HLA I Ab-dependent up-regulation of VCAM-1. Treatment with two carbon monoxide (CO-releasing molecules, which liberate the gaseous HO product CO, blocked HLA I Ab-dependent EC activation. Finally, in an in vitro adhesion assay exposure of ECs to HLA I Abs led to increased monocyte binding, which was counteracted by up-regulation of HO-1. In conclusion, HLA I Ab-dependent EC activation is modulated by endothelial HO-1 and targeted induction of this enzyme may be a novel therapeutic approach for the treatment of AMR in solid organ transplantation.

  15. DNA-dependent protein kinase inhibits AID-induced antibody gene conversion.

    Directory of Open Access Journals (Sweden)

    Adam J L Cook

    2007-04-01

    Full Text Available Affinity maturation and class switching of antibodies requires activation-induced cytidine deaminase (AID-dependent hypermutation of Ig V(DJ rearrangements and Ig S regions, respectively, in activated B cells. AID deaminates deoxycytidine bases in Ig genes, converting them into deoxyuridines. In V(DJ regions, subsequent excision of the deaminated bases by uracil-DNA glycosylase, or by mismatch repair, leads to further point mutation or gene conversion, depending on the species. In Ig S regions, nicking at the abasic sites produced by AID and uracil-DNA glycosylases results in staggered double-strand breaks, whose repair by nonhomologous end joining mediates Ig class switching. We have tested whether nonhomologous end joining also plays a role in V(DJ hypermutation using chicken DT40 cells deficient for Ku70 or the DNA-dependent protein kinase catalytic subunit (DNA-PKcs. Inactivation of the Ku70 or DNA-PKcs genes in DT40 cells elevated the rate of AID-induced gene conversion as much as 5-fold. Furthermore, DNA-PKcs-deficiency appeared to reduce point mutation. The data provide strong evidence that double-strand DNA ends capable of recruiting the DNA-dependent protein kinase complex are important intermediates in Ig V gene conversion.

  16. Vancomycin-induced Immune Thrombocytopenia Proven by the Detection of Vancomycin-dependent Anti-platelet Antibody with Flow Cytometry

    OpenAIRE

    Yamanouchi, Jun; Hato, Takaaki; Shiraishi, Sanshiro; Takeuchi, Kazuto; Yakushijin, Yoshihiro; Yasukawa, Masaki

    2016-01-01

    Vancomycin-induced thrombocytopenia is a rare adverse reaction that may be overlooked because no specific diagnostic test is currently available. We herein report a patient with vancomycin-induced immune thrombocytopenia who was diagnosed by the detection of vancomycin-dependent anti-platelet antibody with flow cytometry. An IgG antibody in the patient's serum reacted with platelets only in the presence of vancomycin. Severe thrombocytopenia gave rise to life-threatening gastrointestinal blee...

  17. Do Clustering Monoclonal Antibody Solutions Really Have a Concentration Dependence of Viscosity?

    Science.gov (United States)

    Pathak, Jai A.; Sologuren, Rumi R.; Narwal, Rojaramani

    2013-01-01

    Protein solution rheology data in the biophysics literature have incompletely identified factors that govern hydrodynamics. Whereas spontaneous protein adsorption at the air/water (A/W) interface increases the apparent viscosity of surfactant-free globular protein solutions, it is demonstrated here that irreversible clusters also increase system viscosity in the zero shear limit. Solution rheology measured with double gap geometry in a stress-controlled rheometer on a surfactant-free Immunoglobulin solution demonstrated that both irreversible clusters and the A/W interface increased the apparent low shear rate viscosity. Interfacial shear rheology data showed that the A/W interface yields, i.e., shows solid-like behavior. The A/W interface contribution was smaller, yet nonnegligible, in double gap compared to cone-plate geometry. Apparent nonmonotonic composition dependence of viscosity at low shear rates due to irreversible (nonequilibrium) clusters was resolved by filtration to recover a monotonically increasing viscosity-concentration curve, as expected. Although smaller equilibrium clusters also existed, their size and effective volume fraction were unaffected by filtration, rendering their contribution to viscosity invariant. Surfactant-free antibody systems containing clusters have complex hydrodynamic response, reflecting distinct bulk and interface-adsorbed protein as well as irreversible cluster contributions. Literature models for solution viscosity lack the appropriate physics to describe the bulk shear viscosity of unstable surfactant-free antibody solutions. PMID:23442970

  18. Antibody drug conjugates and bystander killing: is antigen-dependent internalisation required?

    Science.gov (United States)

    Staudacher, Alexander H; Brown, Michael P

    2017-12-05

    Antibody drug conjugates (ADCs) employ the exquisite specificity of tumour-specific monoclonal antibodies (mAb) for the targeted delivery of highly potent cytotoxic drugs to the tumour site. The chemistry of the linker, which connects the drug to the mAb, determines how and when the drug is released from the mAb. This, as well as the chemistry of the drug, can dictate whether the drug can diffuse into surrounding cells, resulting in 'bystander killing'. Initially, any bystander killing mechanism of action of an ADC was understood to involve an essential sequence of steps beginning with surface antigen targeting, internalisation, intracellular linker cleavage, drug release, and diffusion of drug away from the targeted cell. However, recent studies indicate that, depending on the linker and drug combination, this mechanism may not be essential and ADCs can be cleaved extracellularly or via other mechanisms. In this minireview, we will examine the role of bystander killing by ADCs and explore the emerging evidence of how this can occur independently of internalisation.

  19. Dietary cinnamaldehyde enhances acquisition of specific antibodies following helminth infection in pigs

    DEFF Research Database (Denmark)

    Williams, Andrew R.; Hansen, Tina V. A.; Krych, Lukasz

    2017-01-01

    Dietary phytonutrients such as cinnamaldehyde (CA) may contribute to immune function during pathogen infections, and CA has been reported to have positive effects on gut health when used as feed additive for livestock. Here, we investigated whether CA could enhance antibody production and specific...... immune responses during infection with an enteric pathogen. We examined the effect of dietary CA on plasma antibody levels in parasite-naïve pigs, and subsequently acquisition of humoral immune responses during infection with the parasitic nematode Ascaris suum. Parasite-naïve pigs fed diets supplemented......) and glucose transporter 2 (SLC2A2) in the jejunal mucosa of A.suum-infected pigs. Dietary CA induced only limited changes in the composition of the prokaryotic gut microbiota of A. suum-infected pigs, and in vitro experiments showed that CA did not directly induce proliferation or increase secretion of Ig...

  20. A rational approach to enhancing antibody Fc homodimer formation for robust production of antibody mixture in a single cell line.

    Science.gov (United States)

    Yu, Jie; Wang, Xiaoxiao; Xu, Tao; Jin, Qiuheng; Duan, Jinyuan; Wu, Jie; Wu, Haiyan; Xu, Ting; Ye, Sheng

    2017-10-27

    Combinations of different antibodies have been shown to be more effective for managing certain diseases than monotherapy. Co-expression of the antibody mixture in a single cell line is key to reducing complexity during antibody development and manufacturing. However, co-transfection of multiple light and heavy chains into cells often leads to production of mismatched, heterodimeric by-products that are inactive, making the development of co-expression systems that robustly and efficiently produce highly active antibody mixtures a high priority. In this study, we modified the CH3 domain interface of the antibody fragment crystallizable (Fc) region by changing several charge pairs to create electrostatic interactions favoring Fc homodimer formation and disfavoring Fc heterodimer formation. When co-expressed, these modified antibodies with altered charge polarity across the Fc dimer interface preferentially formed homodimers that fully preserved the functions of each component, rather than inactive heterodimers whose formation was reduced because of rationally designed repulsive interactions. We designed eight different combinations and experimentally screened the best one, which enabled us to produce a binary antibody mixture against the EGF receptor with a minimal heterodimer contaminant. We further determined the crystal structure of a triple-mutated Fc variant in the best combination, and we elucidated the molecular interactions favoring Fc homodimer over heterodimer formation, which provided a structural basis for further optimization. The approach presented here demonstrates the feasibility of rational antibody modification for efficient and consistent production of monoclonal antibody mixtures in a single cell line and thus broadens our options for manufacturing more effective antibody-based therapeutic agents. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. A Cross-Reactive Monoclonal Antibody to Nematode Haemoglobin Enhances Protective Immune Responses to Nippostrongylus brasiliensis

    Science.gov (United States)

    Nieuwenhuizen, Natalie E.; Meter, Jeanne M.; Horsnell, William G.; Hoving, J. Claire; Fick, Lizette; Sharp, Michael F.; Darby, Matthew G.; Parihar, Suraj P.; Brombacher, Frank; Lopata, Andreas L.

    2013-01-01

    Background Nematode secreted haemoglobins have unusually high affinity for oxygen and possess nitric oxide deoxygenase, and catalase activity thought to be important in protection against host immune responses to infection. In this study, we generated a monoclonal antibody (48Eg) against haemoglobin of the nematode Anisakis pegreffii, and aimed to characterize cross-reactivity of 4E8g against haemoglobins of different nematodes and its potential to mediate protective immunity against a murine hookworm infection. Methodology/Principal Findings Immunoprecipitation was used to isolate the 4E8g-binding antigen in Anisakis and Ascaris extracts, which were identified as haemoglobins by peptide mass fingerprinting and MS/MS. Immunological cross-reactivity was also demonstrated with haemoglobin of the rodent hookworm N. brasiliensis. Immunogenicity of nematode haemoglobin in mice and humans was tested by immunoblotting. Anisakis haemoglobin was recognized by IgG and IgE antibodies of Anisakis-infected mice, while Ascaris haemoglobin was recognized by IgG but not IgE antibodies in mouse and human sera. Sequencing of Anisakis haemoglobin revealed high similarity to haemoglobin of a related marine nematode, Psuedoterranova decipiens, which lacks the four –HKEE repeats of Ascaris haemoglobin important in octamer assembly. The localization of haemoglobin in the different parasites was examined by immunohistochemistry and associated with the excretory-secretary ducts in Anisakis, Ascaris and N. brasiliensis. Anisakis haemoglobin was strongly expressed in the L3 stage, unlike Ascaris haemoglobin, which is reportedly mainly expressed in adult worms. Passive immunization of mice with 4E8g prior to infection with N. brasiliensis enhanced protective Th2 immunity and led to a significant decrease in worm burdens. Conclusion The monoclonal antibody 4E8g targets haemoglobin in broadly equivalent anatomical locations in parasitic nematodes and enhances host immunity to a hookworm

  2. Stabilizing the CH2 Domain of an Antibody by Engineering in an Enhanced Aromatic Sequon.

    Science.gov (United States)

    Chen, Wentao; Kong, Leopold; Connelly, Stephen; Dendle, Julia M; Liu, Yu; Wilson, Ian A; Powers, Evan T; Kelly, Jeffery W

    2016-07-15

    Monoclonal antibodies (mAbs) exhibiting highly selective binding to a protein target constitute a large and growing proportion of the therapeutics market. Aggregation of mAbs results in the loss of their therapeutic efficacy and can result in deleterious immune responses. The CH2 domain comprising part of the Fc portion of Immunoglobulin G (IgG) is typically the least stable domain in IgG-type antibodies and therefore influences their aggregation propensity. We stabilized the CH2 domain by engineering an enhanced aromatic sequon (EAS) into the N-glycosylated C'E loop and observed a 4.8 °C increase in the melting temperature of the purified IgG1 Fc fragment. This EAS-stabilized CH2 domain also conferred enhanced stability against thermal and low pH induced aggregation in the context of a full-length monoclonal IgG1 antibody. The crystal structure of the EAS-stabilized (Q295F/Y296A) IgG1 Fc fragment confirms the design principle, i.e., the importance of the GlcNAc1•F295 interaction, and surprisingly reveals that the core fucose attached to GlcNAc1 also engages in an interaction with F295. Inhibition of core fucosylation confirms the contribution of the fucose-Phe interaction to the stabilization. The Q295F/Y296A mutations also modulate the binding affinity of the full-length antibody to Fc receptors by decreasing the binding to low affinity Fc gamma receptors (FcγRIIa, FcγRIIIa, and FcγRIIIb), while maintaining wild-type binding affinity to FcRn and FcγRI. Our results demonstrate that engineering an EAS into the N-glycosylated reverse turn on the C'E loop leads to stabilizing N-glycan-protein interactions in antibodies and that this modification modulates antibody-Fc receptor binding.

  3. HP-1γ Controls High-Affinity Antibody Response to T-Dependent Antigens.

    Science.gov (United States)

    Ha, Ngoc; Pham, Duc-Hung; Shahsafaei, Aliakbar; Naruse, Chie; Asano, Masahide; Thai, To-Ha

    2014-01-01

    In vitro observations suggest a role for the mouse heterochromatin protein 1γ (HP-1γ) in the immune system. However, it has not been shown if and how HP-1γ contributes to immunity in vivo. Here we show that in mice, HP-1γ positively regulates the germinal center reaction and high-affinity antibody response to thymus (T)-dependent antigens by limiting the size of CD8(+) regulatory T-cell (Treg) compartment without affecting progenitor B- or T-cell-development. Moreover, HP-1γ does not control cell proliferation or class switch recombination. Haploinsufficiency of cbx-3 (gene encoding HP-1γ) is sufficient to expand the CD8(+) Treg population and impair the immune response in mice despite the presence of wild-type HP-1α and HP-1β. This is the first in vivo evidence demonstrating the non-redundant role of HP-1γ in immunity.

  4. Novel CagA ELISA exhibits enhanced sensitivity of Helicobacter pylori CagA antibody

    Science.gov (United States)

    Matsuo, Yuichi; Kido, Yasutoshi; Akada, Junko; Shiota, Seiji; Binh, Tran Thanh; Trang, Tran Thi Huyen; Dung, Ho D Q; Tung, Pham Huu; Tri, Tran Dinh; Thuan, Ngo P Minh; Tam, Le Quang; Nam, Bui Chi; Khien, Vu Van; Yamaoka, Yoshio

    2017-01-01

    AIM To develop a novel Helicobacter pylori (H. pylori) CagA antibody enzyme-linked immunosorbent assay (ELISA) suitable for detecting serum anti-CagA antibodies with high sensitivity. METHODS Recombinant East Asian-type CagA protein was purified and immobilized for ELISA. Serum samples from 217 Vietnamese individuals (110 H. pylori-infected and 107 uninfected individuals) were applied. Conventional ELISA from Western-type CagA and our East Asian-type CagA ELISA were evaluated by comparing 38 subjects with the Western-type genotype and 72 subjects with the East Asian-type cagA genotype. Histological scores of the gastric mucosa were determined using the updated Sydney System to examine the relationship with anti-CagA antibody titers. RESULTS Recombinant 70-100 kDa fragments were immobilized on the ELISA plate. In ROC analysis, the area under the curve of our East Asian-type CagA ELISA was comparable to that of conventional CagA ELISA. The sensitivity of the two ELISAs differed depending on the cagA genotype. The sensitivity of East Asian-type CagA ELISA was higher for subjects infected with East Asian-type cagA H. pylori (P pylori (P = 0.056). The titer of anti-CagA antibody tended to correlate with monocyte infiltration scores (r = 0.25, P = 0.058) and was inversely correlated with H. pylori density (r = -0.26, P = 0.043). CONCLUSION The novel ELISA is useful to detect anti-CagA antibodies in East Asian countries, and the titer may be a marker for predicting chronic gastritis. PMID:28104980

  5. Monoclonal antibody against brain calmodulin-dependent protein kinase type II detects putative conformational changes induced by Ca2+-calmodulin

    International Nuclear Information System (INIS)

    LeVine, H. III; Su, J.L.; Sahyoun, N.E.

    1988-01-01

    A mouse monoclonal IgG1 antibody has been generated against the soluble form of the calmodulin-dependent protein kinase type II. This antibody recognizes both the soluble and cytoskeletal forms of the enzyme, requiring Ca 2+ for the interaction. Other divalent cations such as Zn 2+ , Mn 2+ , Cd 2+ , Co 2+ , and Ni 2+ will substitute for Ca 2+ , while Mg 2+ and Ba 2+ will not. The antibody reacts with both the α- and β-subunits on Western blots in a similar Ca 2+ -dependent fashion but with a lower sensitivity. The affinity of the antibody for the kinase is 0.13 nM determined by displacement of 125 I Bolton-Hunter-labeled kinase with unlabeled enzyme. Calmodulin and antibody reciprocally potentiate each other's interaction with the enzyme. This is illustrated both by direct binding studies and by a decrease of the K/sub m app/ for calmodulin and an increase in the V/sub max/ for the autophosphorylation reaction of the enzyme. The antibody thus appears to recognize and stabilize a conformation of the kinase which favors calmodulin binding although it does not itself activate the kinase in the absence of calmodulin. Since the M/sub r/ 30,000 catalytic fragment of the kinase is not immunoreactive, either the antibody combining site of the kinase must be present in the noncatalytic portion of the protein along with the calmodulin binding site or proteolysis interferes with the putative Ca 2+ -dependent conformational change. Thus, monoclonal antibodies can be useful tools in elucidating the mechanism by which Ca 2+ and calmodulin act on the kinase molecule

  6. Contribution of peptide backbone to Anti-citrulline-dependent antibody reactivity

    DEFF Research Database (Denmark)

    Trier, Nicole Hartwig; Dam, Catharina; Olsen, Dorthe

    2015-01-01

    . As ACPAs have been suggested to be involved in the development of RA, knowledge about these antibodies may be crucial. In this study, we examined the influence of peptide backbone for ACPA reactivity in immunoassays. The antibodies were found to be reactive with a central Cit-Gly motif being essential...... is essential for antibody reactivity. Based on these findings it was speculated that any amino acid sequence, which brings the peptide into a properly folded structure for antibody recognition is sufficient for antibody reactivity. These findings are in accordance with the current hypothesis that structural...

  7. Studies on ADCC (antibody-dependent cell-mediated cytotoxicity) using sheep red blood cells as target cells, 2

    International Nuclear Information System (INIS)

    Ichikawa, Yukinobu; Takaya, Masatoshi; Arimori, Shigeru

    1979-01-01

    A non-specific cytotoxic mediator from effector cells (human peripheral blood leukocytes) was investigated in the ADCC (antibody-dependent cell-mediated cytotoxicity) system using antibody-coated sheep red blood cells (SRBC) as target cells. 51 Cr-labelled homologous (sheep) or heterologous (human) red blood cells were used as adjacent cells. Either crude lymphocyte fraction, phagocyte depleted fraction or granulocyte rich fraction separated from human peripheral leukocytes showed moderate cytotoxic effect on homologous adjacent cells, however no cytotoxic activity on heterologous adjacent cells was demonstrated in any leukocyte fraction. This suggests that the cytotoxic effects on homologous adjacent cells were resulted from the translocation of antibody molecules to adjacent cells from antibody-coated target cells. We concluded that the cytotoxic mechanism in this ADCC system was not mediated by non-specific soluble factors released from either human peripheral lymphocytes, monocytes or granulocytes. (author)

  8. HIV-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC) -Mediating Antibodies Decline while NK Cell Function Increases during Antiretroviral Therapy (ART).

    Science.gov (United States)

    Jensen, Sanne Skov; Fomsgaard, Anders; Borggren, Marie; Tingstedt, Jeanette Linnea; Gerstoft, Jan; Kronborg, Gitte; Rasmussen, Line Dahlerup; Pedersen, Court; Karlsson, Ingrid

    2015-01-01

    Understanding alterations in HIV-specific immune responses during antiretroviral therapy (ART), such as antibody-dependent cellular cytotoxicity (ADCC), is important in the development of novel strategies to control HIV-1 infection. This study included 53 HIV-1 positive individuals. We evaluated the ability of effector cells and antibodies to mediate ADCC separately and in combination using the ADCC-PanToxiLux assay. The ability of the peripheral blood mononuclear cells (PBMCs) to mediate ADCC was significantly higher in individuals who had been treated with ART before seroconversion, compared to the individuals initiating ART at a low CD4+ T cell count (ART-naïve individuals. The frequency of CD16 expressing natural killer (NK) cells correlated with both the duration of ART and Granzyme B (GzB) activity. In contrast, the plasma titer of antibodies mediating ADCC declined during ART. These findings suggest improved cytotoxic function of the NK cells if initiating ART early during infection, while the levels of ADCC mediating antibodies declined during ART.

  9. Dose Dependency and a Functional Cutoff for TPO-Antibody Positivity During Pregnancy.

    Science.gov (United States)

    Korevaar, Tim I M; Pop, Victor J; Chaker, Layal; Goddijn, Mariette; de Rijke, Yolanda B; Bisschop, Peter H; Broeren, Maarten A; Jaddoe, Vincent W V; Medici, Marco; Visser, Theo J; Steegers, Eric A P; Vrijkotte, Tanja G; Peeters, Robin P

    2018-02-01

    To investigate a dose dependency of thyroperoxidase antibody (TPOAb) concentrations in relation to thyroid function and premature delivery and define a population-based, pregnancy-specific, functional cutoff for TPOAb positivity. Individual participant meta-analysis of three prospective birth cohorts: the Amsterdam Born Children and their Development study, and the Holistic Approach to Pregnancy. Population-based studies in the Netherlands (2002 to 2014). A total of 11,212 pregnant women (2.5 mU/L (range, 19.4% to 51.3%). Stratified analyses showed that women with TPOAb concentrations below manufacturer cutoffs already had a higher risk of premature delivery, especially when TSH concentrations were high or in the high-normal range. This study demonstrated a dose-dependent relationship between TPOAbs and thyroid function as well as the risk of premature delivery. Furthermore, our results indicate that the currently used cutoffs for TPOAb positivity may be too high. Furthermore, the use of a population-based cutoff for TPOAbs may identify women with a clinically relevant extent of thyroid autoimmunity and a higher risk of premature delivery but that would not be considered TPOAb positive or eligible for treatment otherwise.

  10. Contribution of NK cell education to both direct and anti-HIV-1 antibody-dependent NK cell functions.

    Science.gov (United States)

    Kristensen, Anne B; Kent, Stephen J; Parsons, Matthew S

    2018-03-07

    Antibody Fc-dependent functions are linked to prevention and control of HIV-1 infection. Basic NK cell biology is likely key to understanding the contributions anti-HIV-1 antibody-dependent NK cell activation and cytolysis make to HIV-1 susceptibility and disease progression. The importance of NK cell education through inhibitory receptors specific for self-HLA-I in determining the potency of anti-HIV-1 antibody mediated NK cell activation and cytolysis is controversial. To address this issue more definitively we utilized HLA-I genotyping, flow cytometry staining panels and cytolysis assays to assess the functionality of educated and non-educated peripheral blood NK cells. We now demonstrate that educated NK cells are superior in terms of their capacity to become activated and/or mediate cytolysis following anti-HIV-1 antibody-dependent stimulation. The profiles of activation observed were similar to those observed upon direct stimulation of NK cells with HLA-I devoid target cells. Non-educated NK cells make significantly lower contributions to total NK cell activation than would be expected from their frequency within the total NK cell population (i.e., are hypofunctional) and educated NK cells make similar or higher contributions as their frequency in the total NK cell population. Finally, NK cells educated through at least one killer immunoglobulin-like receptor and NKG2A exhibited the most significant difference between actual and expected contribution to the total NK cell response, based on their frequency within the total NK cell population, suggesting summation of NK cell education through inhibitory receptors determines overall NK cell functionality. These observations have potential implications for understanding HIV-1 vaccine efficacy and disease progression. IMPORTANCE NK cells are major mediators of anti-HIV-1 antibody-dependent functions, including cytokine production and cytolysis. The mechanisms controlling the capacity of individual NK cells to

  11. Fusion of C3d molecule with neutralization epitope(s) of hepatitis E virus enhances antibody avidity maturation and neutralizing activity following DNA immunization.

    Science.gov (United States)

    Yang, Shucai; Wang, Chunling; Fang, Xuefeng; Zhai, Lijie; Dong, Chen; Ding, Lei; Meng, Jihong; Wang, Lixin

    2010-08-01

    Previous studies have identified that a hepatits E virus peptide (HEV-p179), spanning amino acids (aa) 439-617 in the 660-aa protein encoded by open reading frame 2(ORF2) of the Chinese epidemic strain (genotype 4), is the minimal size fragment of conformation-dependent neutralization epitope(s). We report here the successful immunization of mice with DNA vaccines expressing the secreted form of HEV-p179 (fused with a human tissue plasminogen activator (tPA) signal sequence) and the tPA-p179-C3d fusion protein (fused with three tandem copies of the murine complement C3d). Analysis of antibody responses in vaccinated mice revealed that immunizations with tPA-p179-C3d3 DNA vaccine dramatically increased both the level and avidity maturation of antibodies against HEV-p179 compared to p179 and tPA-p179 DNA vaccines. In addition, this increased antibody response correlated with neutralizing titers in a PCR-based cell culture neutralization assay. These results indicate that vaccination with C3d conjugated p179 DNA vaccine enhances antibody responses to HEV, and this approach may be applied to overcome the poor immunogenicity of DNA vaccines to generate HEV neutralizing antibodies. Copyright 2010 Elsevier B.V. All rights reserved.

  12. Chemical Screens Identify Drugs that Enhance or Mitigate Cellular Responses to Antibody-Toxin Fusion Proteins.

    Directory of Open Access Journals (Sweden)

    Antonella Antignani

    Full Text Available The intersection of small molecular weight drugs and antibody-based therapeutics is rarely studied in large scale. Both types of agents are currently part of the cancer armamentarium. However, very little is known about how to combine them in optimal ways. Immunotoxins are antibody-toxin gene fusion proteins engineered to target cancer cells via antibody binding to surface antigens. For fusion proteins derived from Pseudomonas exotoxin (PE, potency relies on the enzymatic domain of the toxin which catalyzes the ADP-ribosylation of EF2 causing inhibition of protein synthesis leading to cell death. Candidate immunotoxins have demonstrated clear value in clinical trials but generally have not been curative as single agents. Therefore we undertook three screens to discover effective combinations that could act synergistically. From the MIPE-3 library of compounds we identified various enhancers of immunotoxin action and at least one major class of inhibitor. Follow-up experiments confirmed the screening data and suggested that immunotoxins when administered with everolimus or nilotinib exhibit favorable combinatory activity and would be candidates for preclinical development. Mechanistic studies revealed that everolimus-immunotoxin combinations acted synergistically on elements of the protein synthetic machinery, including S61 kinase and 4E-BP1 of the mTORC1 pathway. Conversely, PARP inhibitors antagonized immunotoxins and also blocked the toxicity due to native ADP-ribosylating toxins. Thus, our goal of investigating a chemical library was justified based on the identification of several approved compounds that could be developed preclinically as 'enhancers' and at least one class of mitigator to be avoided.

  13. Gold nanoparticles regulate the blimp1/pax5 pathway and enhance antibody secretion in B-cells

    International Nuclear Information System (INIS)

    Lee, Chia-Hui; Syu, Shih-Han; Steven Huang, G; Chen, Yu-Shiun; Chen, Wen Liang; Hussain, Saber M; Aleksandrovich Onischuk, Andrei

    2014-01-01

    Nanoparticles are potential threats to human health and the environment; however, their medical applications as drug carriers targeting cancer cells bring hope to contemporary cancer therapy. As a model drug carrier, gold nanoparticles (GNPs) have been investigated extensively for in vivo toxicity. The effect of GNPs on the immune system, however, has rarely been examined. Antibody-secreting cells were treated with GNPs with diameters ranging from 2 to 50 nm. The GNPs enhanced IgG secretion in a size-dependent manner, with a peak of efficacy at 10 nm. The immune-stimulatory effect reached a maximum at 12 h after treatment but returned to control levels 24 h after treatment. This enhancing effect was validated ex vivo using B-cells isolated from mouse spleen. Evidence from RT-PCR and western blot experiments indicates that GNP-treatment upregulated B-lymphocyte-induced maturation protein 1 (blimp1) and downregulated paired box 5 (pax5). Immunostaining for blimp1 and pax5 in B-cells confirmed that the GNPs stimulated IgG secretion through the blimp1/pax5 pathway. The immunization of mice using peptide-conjugated GNPs indicated that the GNPs were capable of enhancing humoral immunity in a size-dependent manner. This effect was consistent with the bio-distribution of the GNPs in mouse spleen. In conclusion, in vitro, ex vivo, and in vivo evidence supports our hypothesis that GNPs enhance humoral immunity in mouse. The effect on the immune system should be taken into account if nanoparticles are used as carriers for drug delivery. In addition to their toxicity, the immune-stimulatory activity of nanoparticles could play an important role in human health and could have an environmental impact. (paper)

  14. Antitumor activity of chLpMab-2, a human-mouse chimeric cancer-specific antihuman podoplanin antibody, via antibody-dependent cellular cytotoxicity.

    Science.gov (United States)

    Kaneko, Mika K; Yamada, Shinji; Nakamura, Takuro; Abe, Shinji; Nishioka, Yasuhiko; Kunita, Akiko; Fukayama, Masashi; Fujii, Yuki; Ogasawara, Satoshi; Kato, Yukinari

    2017-04-01

    Human podoplanin (hPDPN), a platelet aggregation-inducing transmembrane glycoprotein, is expressed in different types of tumors, and it binds to C-type lectin-like receptor 2 (CLEC-2). The overexpression of hPDPN is involved in invasion and metastasis. Anti-hPDPN monoclonal antibodies (mAbs) such as NZ-1 have shown antitumor and antimetastatic activities by binding to the platelet aggregation-stimulating (PLAG) domain of hPDPN. Recently, we developed a novel mouse anti-hPDPN mAb, LpMab-2, using the cancer-specific mAb (CasMab) technology. In this study we developed chLpMab-2, a human-mouse chimeric anti-hPDPN antibody, derived from LpMab-2. chLpMab-2 was produced using fucosyltransferase 8-knockout (KO) Chinese hamster ovary (CHO)-S cell lines. By flow cytometry, chLpMab-2 reacted with hPDPN-expressing cancer cell lines including glioblastomas, mesotheliomas, and lung cancers. However, it showed low reaction with normal cell lines such as lymphatic endothelial and renal epithelial cells. Moreover, chLpMab-2 exhibited high antibody-dependent cellular cytotoxicity (ADCC) against PDPN-expressing cells, despite its low complement-dependent cytotoxicity. Furthermore, treatment with chLpMab-2 abolished tumor growth in xenograft models of CHO/hPDPN, indicating that chLpMab-2 suppressed tumor development via ADCC. In conclusion, chLpMab-2 could be useful as a novel antibody-based therapy against hPDPN-expressing tumors. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  15. An Fc engineering approach that modulates antibody-dependent cytokine release without altering cell-killing functions.

    Science.gov (United States)

    Kinder, Michelle; Greenplate, Allison R; Strohl, William R; Jordan, Robert E; Brezski, Randall J

    2015-01-01

    Cytotoxic therapeutic monoclonal antibodies (mAbs) often mediate target cell-killing by eliciting immune effector functions via Fc region interactions with cellular and humoral components of the immune system. Key functions include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). However, there has been increased appreciation that along with cell-killing functions, the induction of antibody-dependent cytokine release (ADCR) can also influence disease microenvironments and therapeutic outcomes. Historically, most Fc engineering approaches have been aimed toward modulating ADCC, ADCP, or CDC. In the present study, we describe an Fc engineering approach that, while not resulting in impaired ADCC or ADCP, profoundly affects ADCR. As such, when peripheral blood mononuclear cells are used as effector cells against mAb-opsonized tumor cells, the described mAb variants elicit a similar profile and quantity of cytokines as IgG1. In contrast, although the variants elicit similar levels of tumor cell-killing as IgG1 with macrophage effector cells, the variants do not elicit macrophage-mediated ADCR against mAb-opsonized tumor cells. This study demonstrates that Fc engineering approaches can be employed to uncouple macrophage-mediated phagocytic and subsequent cell-killing functions from cytokine release.

  16. Malignant monoblasts can function as effector cells in natural killer cell and antibody-dependent cellular cytotoxicity assays

    DEFF Research Database (Denmark)

    Hokland, P; Hokland, M; Ellegaard, J

    1981-01-01

    This is the first report describing natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) of malignant monoblasts. Pure acute monoblastic leukemia was diagnosed in bone marrow aspirations from two patients by use of conventional cytochemical methods as well as multiple immunologic...

  17. Overnight Sleep Enhances Hippocampus-Dependent Aspects of Spatial Memory.

    Science.gov (United States)

    Nguyen, Nam D; Tucker, Matthew A; Stickgold, Robert; Wamsley, Erin J

    2013-07-01

    Several studies have now demonstrated that spatial information is processed during sleep, and that posttraining sleep is beneficial for human navigation. However, it remains unclear whether the effects of sleep are primarily due to consolidation of cognitive maps, or alternatively, whether sleep might also affect nonhippocampal aspects of navigation (e.g., speed of motion) involved in moving through a virtual environment. Participants were trained on a virtual maze navigation task (VMT) and then given a memory test following either a day of wakefulness or a night of sleep. Subjects reported to the laboratory for training at either 10:00am or 10:00pm, depending on randomly assigned condition, and were tested 11 h later. Overnight subjects slept in the laboratory with polysomnography. A hospital-based academic sleep laboratory. Thirty healthy college student volunteers. N/A. Point-by-point position data were collected from the VMT. Analysis of the movement data revealed a sleep-dependent improvement in maze completion time (P sleep benefitted performance, not because subjects moved faster through the maze, but because they were more accurate in navigating to the goal. These findings suggest that sleep enhances participants' knowledge of the spatial layout of the maze, contributing to the consolidation of hippocampus-dependent spatial information. Nguyen ND; Tucker MA; Stickgold R; Wamsley EJ. Overnight sleep enhances hippocampus-dependent aspects of spatial memory. SLEEP 2013;36(7):1051-1057.

  18. Early and enhanced antitoxin responses elicited with complexes of tetanus toxoid and specific mouse and human antibodies

    International Nuclear Information System (INIS)

    Stoner, R.D.; Terres, G.; Hess, M.W.

    1975-01-01

    Primary tetanus antitoxin responses were early and enhanced in mice when tetanus toxoid was administered in complex with specific isologous antitoxin or specific mouse γ-globulin. Antitoxin responses were enhanced when fluid tetanus toxoid was complexed in vitro in antigen-to-antibody ratios of equivalence or antigen excess; responses to complexed toxoid in antibody excess were comparatively repressed. Primary responses were greatly inhibited in mice immunized with the same amount of toxoid complexed at equivalence or in antibody excess with specific human γ-globulin. Although primary responses were totally repressed, a primed state developed; a second injection of fluid toxoid within a few days produced excellent antitoxin responses. Separate injections of antigen and antibody at different sites produced an excellent in vivo primed state for early and high responses. Antibody production after stimulation with complexed toxoid was also enhanced in mice irradiated with 400 rads, a dose that ordinarily completely suppresses primary responses with fluid toxoid alone. These data provide evidence for the efficacy of antigen-antibody complexes in early and active immunization. (U.S.)

  19. Enhanced opsonisation of Rhesus D-positive human red blood cells by recombinant polymeric immunoglobulin G anti-G antibodies.

    Science.gov (United States)

    Díaz-Solano, Dylana; Fuenmayor, Jaheli; Montaño, Ramon F

    2018-02-01

    Anti-RhD antibodies (anti-D) are important in the prophylaxis of haemolytic disease of the foetus and newborn (HDFN) due to RhD incompatibility. Current preparations of anti-D are sourced from hyperimmune human plasma, so its production carries a risk of disease and is dependent on donor availability. Despite the efforts to develop a monoclonal preparation with similar prophylactic properties to the plasma-derived anti-D, no such antibody is yet available. Here we studied the agglutinating, opsonic and haemolytic activities of two recombinant polymeric immunoglobulins (Ig) against the G antigen of the Rh complex. Recombinant polymeric anti-G IgG1 (IgG1μtp) and IgG3 (IgG3μtp) were produced in vitro, purified by protein G-affinity chromatography, and analysed by gel electrophoresis. Their agglutinating, opsonic and haemolytic activities were evaluated using haemagglutination, erythrophagocytosis, and complement activation assays. The recombinant IgG1μtp and IgG3μtp anti-G antibodies ranged from 150,000 to 1,000,000 Da in molecular weight, indicating the formation of polymeric IgG. No complement activation or haemolytic activity was detected upon incubation of RhD-positive red-blood cells with the polymeric anti-G IgG. Both polymers were better opsonins than a prophylactic preparation of plasma-derived anti-D. The enhanced opsonic properties of the polymeric anti-G IgG1μtp and IgG3μtp could allow them to mediate the clearance of RhD-positive red blood cells from circulation more efficiently than natural or other synthetic prophylactic anti-D options. Their inability to induce complement-mediated haemolysis would be prophylactically convenient and is comparable in vitro to that of the available plasma-derived polyclonal anti-D preparations. The described properties suggest that polymeric antibodies like these (but with anti-D specificity) may be testable candidates for prophylaxis of HDFN caused by anti-D.

  20. Glycoengineering of therapeutic antibodies enhances monocyte/macrophage-mediated phagocytosis and cytotoxicity.

    Science.gov (United States)

    Herter, Sylvia; Birk, Martina C; Klein, Christian; Gerdes, Christian; Umana, Pablo; Bacac, Marina

    2014-03-01

    Therapeutic Abs possess several clinically relevant mechanisms of action including perturbation of tumor cell signaling, activation of complement-dependent cytotoxicity, Ab-dependent cellular cytotoxicity (ADCC), Ab-dependent cellular phagocytosis (ADCP), and induction of adaptive immunity. In view of the important role of phagocytic lineage cells in the mechanism of action of therapeutic Abs, we analyzed FcγR receptor-dependent effector functions of monocytes and macrophages triggered by glycoengineered (GE) Abs (having enhanced FcγRIIIa [CD16a] binding affinity) versus their wild-type (WT) counterparts under different experimental conditions. We first defined the precise FcγR repertoire on classical and nonclassical intermediate monocytes--M1 and M2c macrophage populations. We further show that WT and GE Abs display comparable binding and induce similar effector functions (ADCC and ADCP) in the absence of nonspecific, endogenous IgGs. However, in the presence of these IgGs (i.e., in a situation that more closely mimics physiologic conditions), GE Abs display significantly superior binding and promote stronger monocyte and macrophage activity. These data show that in addition to enhancing CD16a-dependent NK cell cytotoxicity, glycoengineering also enhances monocyte and macrophage phagocytic and cytotoxic activities through enhanced binding to CD16a under conditions that more closely resemble the physiologic setting.

  1. Instrumental learning: an animal model for sleep dependent memory enhancement.

    Science.gov (United States)

    Leenaars, Cathalijn H C; Girardi, Carlos E N; Joosten, Ruud N J M A; Lako, Irene M; Ruimschotel, Emma; Hanegraaf, Maaike A J; Dematteis, Maurice; Feenstra, Matthijs G P; Van Someren, Eus J W

    2013-07-15

    The relationship between learning and sleep is multifaceted; learning influences subsequent sleep characteristics, which may in turn influence subsequent memory. Studies in humans indicate that sleep may not only prevent degradation of acquired memories, but even enhance performance without further practice. In a rodent instrumental learning task, individual differences occur in how fast rats learn to associate lever pressing with food reward. Rats habitually sleep between learning sessions, and may differ in this respect. The current study assessed if the instrumental leaning paradigm could serve as a model to study sleep-dependent memory enhancement. Male Wistar rats performed 2 sessions of instrumental learning per day for 1-3 days. Electroencephalography was recorded both before and after the sessions. Sleep deprivation (3 h) was applied between the first and second session in a subgroup of rats. Measurements comprised the number of lever presses in each session, slow wave sleep (SWS) duration, Rapid Eye Movement Sleep (REMS) duration and sleep spindles. Baseline sleep parameters were similar for fast and slow learning rats. Task-exposure increased REMS-duration. The increase in REMS-duration was observed specifically after sessions in which learning occurred, but not after a later session. Sleep deprivation during the 3h period between the initial two sessions interfered with performance enhancement, but did not prevent this in all rats. Our considered movement control protocol induced partial sleep deprivation and also interfered with performance enhancement. The classic instrumental learning task provides a practical model for animal studies on sleep-dependent memory enhancement. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. ChLpMab-23: Cancer-Specific Human-Mouse Chimeric Anti-Podoplanin Antibody Exhibits Antitumor Activity via Antibody-Dependent Cellular Cytotoxicity.

    Science.gov (United States)

    Kaneko, Mika K; Nakamura, Takuro; Kunita, Akiko; Fukayama, Masashi; Abe, Shinji; Nishioka, Yasuhiko; Yamada, Shinji; Yanaka, Miyuki; Saidoh, Noriko; Yoshida, Kanae; Fujii, Yuki; Ogasawara, Satoshi; Kato, Yukinari

    2017-06-01

    Podoplanin is expressed in many cancers, including oral cancers and brain tumors. The interaction between podoplanin and its receptor C-type lectin-like receptor 2 (CLEC-2) has been reported to be involved in cancer metastasis and tumor malignancy. We previously established many monoclonal antibodies (mAbs) against human podoplanin using the cancer-specific mAb (CasMab) technology. LpMab-23 (IgG 1 , kappa), one of the mouse anti-podoplanin mAbs, was shown to be a CasMab. However, we have not shown the usefulness of LpMab-23 for antibody therapy against podoplanin-expressing cancers. In this study, we first determined the minimum epitope of LpMab-23 and revealed that Gly54-Leu64 peptide, especially Gly54, Thr55, Ser56, Glu57, Asp58, Arg59, Tyr60, and Leu64 of podoplanin, is a critical epitope of LpMab-23. We further produced human-mouse chimeric LpMab-23 (chLpMab-23) and investigated whether chLpMab-23 exerts antibody-dependent cellular cytotoxicity (ADCC) and antitumor activity. In flow cytometry, chLpMab-23 showed high sensitivity against a podoplanin-expressing glioblastoma cell line, LN319, and an oral cancer cell line, HSC-2. chLpMab-23 also showed ADCC activity against podoplanin-expressing CHO cells (CHO/podoplanin). In xenograft models with HSC-2 and CHO/podoplanin, chLpMab-23 exerts antitumor activity using human natural killer cells, indicating that chLpMab-23 could be useful for antibody therapy against podoplanin-expressing cancers.

  3. Monoclonal antibodies against peptidorhamnomannans of Scedosporium apiospermum enhance the pathogenicity of the fungus.

    Directory of Open Access Journals (Sweden)

    Livia C L Lopes

    2010-10-01

    Full Text Available Scedosporium apiospermum is part of the Pseudallescheria-Scedosporium complex. Peptidorhamnomannans (PRMs are cell wall glycopeptides present in some fungi, and their structures have been characterized in S. apiospermum, S. prolificans and Sporothrix schenckii. Prior work shows that PRMs can interact with host cells and that the glycopeptides are antigenic. In the present study, three monoclonal antibodies (mAbs, IgG1 to S. apiospermum derived PRM were generated and their effects on S. apiospermum were examined in vitro and in vivo. The mAbs recognized a carbohydrate epitope on PRM. In culture, addition of the PRM mAbs increased S. apiospermum conidia germination and reduced conidial phagocytosis by J774.16 macrophages. In a murine infection model, mice treated with antibodies to PRM died prior to control animals. Thus, PRM is involved in morphogenesis and the binding of this glycopeptide by mAbs enhanced the virulence of the fungus. Further insights into the effects of these glycopeptides on the pathobiology of S. apiospermum may lead to new avenues for preventing and treating scedosporiosis.

  4. Microshell Arrays Enhanced Sensitivity in Detection of Specific Antibody for Reduced Graphene Oxide Optical Sensor

    Directory of Open Access Journals (Sweden)

    Wen-Shuai Jiang

    2017-01-01

    Full Text Available Protein-protein interactions play an important role in the investigation of biomolecules. In this paper, we reported on the use of a reduced graphene oxide microshell (RGOM-based optical biosensor for the determination of goat anti-rabbit IgG. The biosensor was prepared through a self-assembly of monolayers of monodisperse polystyrene microspheres, combined with a high-temperature reduction, in order to decorate the RGOM with rabbit IgG. The periodic microshells allowed a simpler functionalization and modification of RGOM with bioreceptor units, than reduced graphene oxide (RGO. With additional antibody-antigen binding, the RGOM-based biosensor achieved better real-time and label-free detection. The RGOM-based biosensor presented a more satisfactory response to goat anti-rabbit IgG than the RGO-based biosensor. This method is promising for immobilizing biomolecules on graphene surfaces and for the fabrication of biosensors with enhanced sensitivity.

  5. C-reactive protein enhances the respiratory burst of neutrophils-induced by antineutrophil cytoplasmic antibody.

    Science.gov (United States)

    Xu, Peng-cheng; Hao, Jian; Yang, Xiao-wei; Chang, Dong-yuan; Chen, Min; Zhao, Ming-hui

    2012-10-01

    Serum C-reactive protein (CRP) was one of the useful biomarkers for evaluating the disease activity in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Cumulating studies proved that CRP was pathogenic in a variety of diseases. In the current study, the in vitro effects of CRP to prime neutrophils for ANCA-induced respiratory burst were investigated with flow cytometry. Without TNF-α in the reactive system, ANCA could only induce a slight level of respiratory burst of neutrophils. CRP could enhance the respiratory burst of neutrophils induced by ANCA against myeloperoxidse [mean fluorescence intensity (MFI, 68.45 ± 16.87 vs. 58.65 ± 15.09, P Heat-treated CRP could not enhance the levels of neutrophils respiratory burst induced by ANCA or increase the expression of membrane proteinase 3 of neutrophils. So CRP can prime neutrophils and enhance the respiratory burst induced by ANCA and might be pathogenic in AAV. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. [IgG anti HSV I antibodies in saliva in a drug dependent population].

    Science.gov (United States)

    Urquía, M; Ceballos, A; Ruiz, M; Pérez-Alférez, N

    1989-11-01

    It is known the presence of receptors for the morphine products on the Lymphocytes B. These receptors could have a modulator action on the production of antibodies in drug addicts. We have determined the existence of IgG anti-HSV I in saliva and serum in a heroin population. Our results show that here is not significant differences between the heroin population and the control group, when the antibodies are detected in serum. But significant differences do exist between the number of people with antibodies anti-HSV I in saliva.

  7. HIV-1 Env- and Vpu-Specific Antibody-Dependent Cellular Cytotoxicity Responses Associated with Elite Control of HIV.

    Science.gov (United States)

    Madhavi, Vijaya; Wines, Bruce D; Amin, Janaki; Emery, Sean; Lopez, Ester; Kelleher, Anthony; Center, Rob J; Hogarth, P Mark; Chung, Amy W; Kent, Stephen J; Stratov, Ivan

    2017-09-15

    Studying HIV-infected individuals who control HIV replication (elite controllers [ECs]) enables exploration of effective anti-HIV immunity. HIV Env-specific and non-Env-specific antibody-dependent cellular cytotoxicity (ADCC) may contribute to protection from progressive HIV infection, but the evidence is limited. We recruited 22 ECs and matched them with 44 viremic subjects. HIV Env- and Vpu-specific ADCC responses in sera were studied using a novel enzyme-linked immunosorbent assay (ELISA)-based dimeric recombinant soluble FcγRIIIa (rsFcγRIIIa)-binding assay, surface plasmon resonance, antibody-dependent natural killer (NK) cell activation assays, and ADCC-mediated killing assays. ECs had higher levels of HIV Env-specific antibodies capable of binding FcγRIIIa, activating NK cells, and mediating granzyme B activity (all P Env-specific and Vpu epitope-specific ADCC in effective immune responses against HIV among ECs. Our findings have implications for understanding the role of ADCC in HIV control. IMPORTANCE Understanding immune responses associated with elite control of HIV may aid the development of immunotherapeutic and vaccine strategies for controlling HIV infection. Env is a major HIV protein target of functional antibody responses that are heightened in ECs. Interestingly, EC antibodies also target Vpu, an accessory protein crucial to HIV, which degrades CD4 and antagonizes tetherin. Antibodies specific to Vpu are a common feature of the immune response of ECs that may prove to be of functional importance to the design of improved ADCC-based immunotherapy and preventative HIV vaccines. Copyright © 2017 American Society for Microbiology.

  8. Content dependent selection of image enhancement parameters for mobile displays

    Science.gov (United States)

    Lee, Yoon-Gyoo; Kang, Yoo-Jin; Kim, Han-Eol; Kim, Ka-Hee; Kim, Choon-Woo

    2011-01-01

    Mobile devices such as cellular phones and portable multimedia player with capability of playing terrestrial digital multimedia broadcasting (T-DMB) contents have been introduced into consumer market. In this paper, content dependent image quality enhancement method for sharpness and colorfulness and noise reduction is presented to improve perceived image quality on mobile displays. Human visual experiments are performed to analyze viewers' preference. Relationship between the objective measures and the optimal values of image control parameters are modeled by simple lookup tables based on the results of human visual experiments. Content dependent values of image control parameters are determined based on the calculated measures and predetermined lookup tables. Experimental results indicate that dynamic selection of image control parameters yields better image quality.

  9. Noninfectious retrovirus particles drive the APOBEC3/Rfv3 dependent neutralizing antibody response.

    Directory of Open Access Journals (Sweden)

    Diana S Smith

    2011-10-01

    Full Text Available Members of the APOBEC3 family of deoxycytidine deaminases counteract a broad range of retroviruses in vitro through an indirect mechanism that requires virion incorporation and inhibition of reverse transcription and/or hypermutation of minus strand transcripts in the next target cell. The selective advantage to the host of this indirect restriction mechanism remains unclear, but valuable insights may be gained by studying APOBEC3 function in vivo. Apobec3 was previously shown to encode Rfv3, a classical resistance gene that controls the recovery of mice from pathogenic Friend retrovirus (FV infection by promoting a more potent neutralizing antibody (NAb response. The underlying mechanism does not involve a direct effect of Apobec3 on B cell function. Here we show that while Apobec3 decreased titers of infectious virus during acute FV infection, plasma viral RNA loads were maintained, indicating substantial release of noninfectious particles in vivo. The lack of plasma virion infectivity was associated with a significant post-entry block during early reverse transcription rather than G-to-A hypermutation. The Apobec3-dependent NAb response correlated with IgG binding titers against native, but not detergent-lysed virions. These findings indicate that innate Apobec3 restriction promotes NAb responses by maintaining high concentrations of virions with native B cell epitopes, but in the context of low virion infectivity. Finally, Apobec3 restriction was found to be saturable in vivo, since increasing FV inoculum doses resulted in decreased Apobec3 inhibition. By analogy, maximizing the release of noninfectious particles by modulating APOBEC3 expression may improve humoral immunity against pathogenic human retroviral infections.

  10. Reynolds-number dependence of turbulence enhancement on collision growth

    Directory of Open Access Journals (Sweden)

    R. Onishi

    2016-10-01

    Full Text Available This study investigates the Reynolds-number dependence of turbulence enhancement on the collision growth of cloud droplets. The Onishi turbulent coagulation kernel proposed in Onishi et al. (2015 is updated by using the direct numerical simulation (DNS results for the Taylor-microscale-based Reynolds number (Reλ up to 1140. The DNS results for particles with a small Stokes number (St show a consistent Reynolds-number dependence of the so-called clustering effect with the locality theory proposed by Onishi et al. (2015. It is confirmed that the present Onishi kernel is more robust for a wider St range and has better agreement with the Reynolds-number dependence shown by the DNS results. The present Onishi kernel is then compared with the Ayala–Wang kernel (Ayala et al., 2008a; Wang et al., 2008. At low and moderate Reynolds numbers, both kernels show similar values except for r2 ∼ r1, for which the Ayala–Wang kernel shows much larger values due to its large turbulence enhancement on collision efficiency. A large difference is observed for the Reynolds-number dependences between the two kernels. The Ayala–Wang kernel increases for the autoconversion region (r1, r2 < 40 µm and for the accretion region (r1 < 40 and r2 > 40 µm; r1 > 40 and r2 < 40 µm as Reλ increases. In contrast, the Onishi kernel decreases for the autoconversion region and increases for the rain–rain self-collection region (r1, r2 > 40 µm. Stochastic collision–coalescence equation (SCE simulations are also conducted to investigate the turbulence enhancement on particle size evolutions. The SCE with the Ayala–Wang kernel (SCE-Ayala and that with the present Onishi kernel (SCE-Onishi are compared with results from the Lagrangian Cloud Simulator (LCS; Onishi et al., 2015, which tracks individual particle motions and size evolutions in homogeneous isotropic turbulence. The SCE-Ayala and SCE-Onishi kernels show consistent

  11. Chimeric Anti-Human Podoplanin Antibody NZ-12 of Lambda Light Chain Exerts Higher Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity Compared with NZ-8 of Kappa Light Chain.

    Science.gov (United States)

    Kaneko, Mika K; Abe, Shinji; Ogasawara, Satoshi; Fujii, Yuki; Yamada, Shinji; Murata, Takeshi; Uchida, Hiroaki; Tahara, Hideaki; Nishioka, Yasuhiko; Kato, Yukinari

    2017-02-01

    Podoplanin (PDPN), a type I transmembrane 36-kDa glycoprotein, is expressed not only in normal cells, such as renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, but also in cancer cells, including brain tumors and lung squamous cell carcinomas. Podoplanin activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and the podoplanin/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced neutralizing anti-human podoplanin monoclonal antibody (mAb), clone NZ-1 (rat IgG 2a , lambda), which neutralizes the podoplanin/CLEC-2 interaction and inhibits platelet aggregation and cancer metastasis. Human-rat chimeric antibody, NZ-8, was previously developed using variable regions of NZ-1 and human constant regions of heavy chain (IgG 1 ) and light chain (kappa chain). Although NZ-8 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cells, the binding affinity of NZ-8 was lower than that of NZ-1. Herein, we produced a novel human-rat chimeric antibody, NZ-12, the constant regions of which consist of IgG 1 heavy chain and lambda light chain. Using flow cytometry, we demonstrated that the binding affinity of NZ-12 was much higher than that of NZ-8. Furthermore, ADCC and CDC activities of NZ-12 were significantly increased against glioblastoma cell lines (LN319 and D397) and lung cancer cell line (PC-10). These results suggested that NZ-12 could become a promising therapeutic antibody against podoplanin-expressing brain tumors and lung cancers.

  12. Antibodies targeting dengue virus envelope domain III are not required for serotype-specific protection or prevention of enhancement in vivo

    OpenAIRE

    Williams, Katherine L.; Wahala, Wahala M.P.B.; Orozco, Susana; de Silva, Aravinda M.; Harris, Eva

    2012-01-01

    The envelope (E) protein of dengue virus (DENV) is composed of three domains (EDI, EDII, EDIII) and is the main target of neutralizing antibodies. Many monoclonal antibodies that bind EDIII strongly neutralize DENV. However in vitro studies indicate that anti-EDIII antibodies contribute little to the neutralizing potency of human DENV-immune serum. In this study, we assess the role of anti-EDIII antibodies in mouse and human DENV-immune serum in neutralizing or enhancing DENV infection in mic...

  13. Pre-existing neutralizing antibody mitigates B cell dysregulation and enhances the Env-specific antibody response in SHIV-infected rhesus macaques.

    Directory of Open Access Journals (Sweden)

    Juan Pablo Jaworski

    Full Text Available Our central hypothesis is that protection against HIV infection will be powerfully influenced by the magnitude and quality of the B cell response. Although sterilizing immunity, mediated by pre-formed abundant and potent antibodies is the ultimate goal for B cell-targeted HIV vaccine strategies, scenarios that fall short of this may still confer beneficial defenses against viremia and disease progression. We evaluated the impact of sub-sterilizing pre-existing neutralizing antibody on the B cell response to SHIV infection. Adult male rhesus macaques received passive transfer of a sub-sterilizing amount of polyclonal neutralizing immunoglobulin (Ig purified from previously infected animals (SHIVIG or control Ig prior to intra-rectal challenge with SHIVSF162P4 and extensive longitudinal sampling was performed. SHIVIG treated animals exhibited significantly reduced viral load and increased de novo Env-specific plasma antibody. Dysregulation of the B cell profile was grossly apparent soon after infection in untreated animals; exemplified by a ≈50% decrease in total B cells in the blood evident 2-3 weeks post-infection which was not apparent in SHIVIG treated animals. IgD+CD5+CD21+ B cells phenotypically similar to marginal zone-like B cells were highly sensitive to SHIV infection, becoming significantly decreased as early as 3 days post-infection in control animals, while being maintained in SHIVIG treated animals, and were highly correlated with the induction of Env-specific plasma antibody. These results suggest that B cell dysregulation during the early stages of infection likely contributes to suboptimal Env-specific B cell and antibody responses, and strategies that limit this dysregulation may enhance the host's ability to eliminate HIV.

  14. Understanding Transcriptional Enhancement in Monoclonal Antibody-Producing Chinese Hamster Ovary Cells

    Science.gov (United States)

    Nicoletti, Sarah E.

    With the demand for monoclonal antibody (mAB) therapeutics continually increasing, the need to better understand what makes a high productivity clone has gained substantial interest. Monoclonal antibody producing Chinese hamster ovary (CHO) cells with different productivities were provided by a biopharmaceutical company for investigation. Gene copy numbers, mRNA levels, and mAb productivities were previously determined for two low producing clones and their amplified progeny. These results showed an increase in mRNA copy number in amplified clones, which correlated to the observed increases in specific productivity of these clones. The presence of multiple copies of mRNA per one copy of DNA in the higher productivity clones has been coined as transcriptional enhancement. The methylation status of the CMV promoter as well as transcription factor/promoter interactions were evaluated to determine the cause of transcriptional enhancement. Methylation analysis via bisulfite sequencing revealed no significant difference in overall methylation status of the CMV promoter. These data did, however, reveal the possibility of differential interactions of transcription factors between the high and low productivity cell clones. This finding was further supported by chromatin immunoprecipitations previously performed in the lab, as well as literature studies. Transcription activator-like effector (TALE) binding proteins were constructed and utilized to selectively immunoprecipitate the CMV promoter along with its associated transcription factors in the different CHO cell clones. Cells were transfected with the TALE proteins, harvested and subjected to a ChIP-like procedure. Results obtained from the TALE ChIP demonstrated the lack of binding of the protein to the promoter and the need to redesign the TALE. Overall, results obtained from this study were unable to give a clear indication as to the causes of transcriptional enhancement in the amplified CHO cell clones. Further

  15. Outer membrane protein complex of Meningococcus enhances the antipolysaccharide antibody response to pneumococcal polysaccharide-CRM₁₉₇ conjugate vaccine.

    Science.gov (United States)

    Lai, Zengzu; Schreiber, John R

    2011-05-01

    Bacterial polysaccharides (PS) are T cell-independent antigens that do not induce immunologic memory and are poor immunogens in infants. Conjugate vaccines in which the PS is covalently linked to a carrier protein have enhanced immunogenicity that resembles that of T cell-dependent antigens. The Haemophilus influenzae type b (Hib) conjugate vaccine, which uses the outer membrane protein complex (OMPC) from meningococcus as a carrier protein, elicits protective levels of anti-capsular PS antibody (Ab) after a single dose, in contrast to other conjugate vaccines, which require multiple doses. We have previously shown that OMPC robustly engages Toll-like receptor 2 (TLR2) and enhances the early anti-Hib PS Ab titer associated with an increase in TLR2-mediated induction of cytokines. We now show that the addition of OMPC to the 7-valent pneumococcal PS-CRM₁₉₇ conjugate vaccine during immunization significantly increases the anti-PS IgG and IgM responses to most serotypes of pneumococcus contained in the vaccine. The addition of OMPC also increased the likelihood of anti-PS IgG3 production against serotypes 4, 6B, 9V, 18C, 19F, and 23F. Splenocytes from mice who had received OMPC with the pneumococcal conjugate vaccine produced significantly more interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) than splenocytes from mice who received phosphate-buffered saline (PBS) plus the conjugate vaccine. We conclude that OMPC enhances the anti-PS Ab response to pneumococcal PS-CRM₁₉₇ conjugate vaccine, an effect associated with a distinct change in cytokine profile. It may be possible to reduce the number of conjugate vaccine doses required to achieve protective Ab levels by priming with adjuvants that are TLR2 ligands.

  16. Oxidative challenge enhances REGγ-proteasome-dependent protein degradation.

    Science.gov (United States)

    Zhang, Yuanyuan; Liu, Shuang; Zuo, Qiuhong; Wu, Lin; Ji, Lei; Zhai, Wanli; Xiao, Jianru; Chen, Jiwu; Li, Xiaotao

    2015-05-01

    Elimination of oxidized proteins is important to cells as accumulation of damaged proteins causes cellular dysfunction, disease, and aging. Abundant evidence shows that the 20S proteasome is largely responsible for degradation of oxidative proteins in both ubiquitin-dependent and ubiquitin-independent pathways. However, the role of the REGγ-proteasome in degrading oxidative proteins remains unclear. Here, we focus on two of the well-known REGγ-proteasome substrates, p21(Waf1/Cip1) and hepatitis C virus (HCV) core protein, to analyze the impact of oxidative stress on REGγ-proteasome functions. We demonstrate that REGγ-proteasome is essential for oxidative stress-induced rapid degradation of p21 and HCV proteins. Silencing REGγ abrogated this response in multiple cell lines. Furthermore, pretreatment with proteasome inhibitor MG132 completely blunted oxidant-induced p21 degradation, indicating a proteasome-dependent action. Cellular oxidation promoted REGγ-proteasome-dependent trypsin-like activity by enhancing the interaction between REGγ and 20S proteasome. Antioxidant could counteract oxidation-induced protein degradation, indicating that REGγ-proteasome activity may be regulated by redox state. This study provides further insights into the actions of a unique proteasome pathway in response to an oxidative stress environment, implying a novel molecular basis for REGγ-proteasome functions in antioxidation. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Enhanced jejunal production of antibodies to Klebsiella and other Enterobacteria in patients with ankylosing spondylitis and rheumatoid arthritis.

    Science.gov (United States)

    Mäki-Ikola, O; Hällgren, R; Kanerud, L; Feltelius, N; Knutsson, L; Granfors, K

    1997-07-01

    To measure gut immunity directly in jejunal fluid in patients with ankylosing spondylitis (AS) and rheumatoid arthritis (RA). Antibodies against three different Enterobacterias were measured in jejunal perfusion fluids (collected by a double balloon perfusion device) of 19 patients with AS, 14 patients with RA, and 22 healthy controls using enzyme linked immunosorbent assay. The AS patients had significantly increased jejunal fluid concentrations of IgM, IgG, and IgA class antibodies against Klebsiella pneumoniae, and IgM and IgA class antibodies against Escherichia coli and Proteus mirabilis compared with healthy controls. When compared with the patients with RA, the AS patients had higher concentrations of IgA and IgG class antibodies only against K pneumoniae. The RA patients had higher IgM class antibody concentrations against all three studied Enterobacterias, when compared with the healthy controls, suggesting an enhanced mucosal immune response in these patients. A three month treatment with sulphasalazine did not decrease enterobacterial antibody concentrations in the 10 patients with AS. There is strong direct evidence for an abnormal mucosal humoral immune response particularly to K pneumoniae in patients with AS.

  18. Enhancement of retroviral infection in vitro by anti-Le(y) IgG: reversal by humanization of monoclonal mouse antibody

    DEFF Research Database (Denmark)

    Hansen, J E; Sørensen, A M; Arendrup, M

    1993-01-01

    Monoclonal mouse IgG3 antibody (ABL 364) against the carbohydrate Le(y) antigen enhanced infection in vitro with HTLV-1 and with HIV-1 when propagated in both transformed and normal lymphocytes. Enhancement was independent of complement, occurred with both lymphocytes and monocytes as target cells......, and did not use either L(ey) epitopes on target cells for cross-linkage of virus to the cell or the Fc part of the antibody as a ligand for any cellular receptor. For enhancement to occur, binding of anti-Le(y) antibody to virus was required to take place before virus binding to its specific receptor...... also enhanced infection, a human/mouse chimeric antibody and a fully humanized antibody had no enhancing effect on free virus infection. We suggest that binding of anti-Le(y) ABL 364 or its F(ab)2 fragment induced a conformational change in the gp120 oligomers facilitating the process of infection...

  19. Complement-dependent pathogenicity of brain-specific antibodies in cerebrospinal fluid

    DEFF Research Database (Denmark)

    Asgari, Nasrin; Khorooshi, Reza; Lillevang, Søren T

    2013-01-01

    The specificity and potential pathogenicity of autoantibodies vary between neurological diseases. It is often unclear whether their detection in cerebrospinal fluid (CSF) is a consequence or a cause of pathology. The goal was to test whether administration of brain-specific antibodies into CSF...

  20. Aberrantly glycosylated MUC1 is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity

    DEFF Research Database (Denmark)

    Lavrsen, Kirstine; Madsen, Caroline B; Rasch, Morten G

    2013-01-01

    not covered by immunological tolerance in MUC1 humanized mice and man. The objective of this study was to determine if mouse antibodies to this Tn-MUC1 epitope induce antibody-dependent cellular cytotoxicity (ADCC) pivotal for their potential use in cancer immunotherapy. Binding affinity of mAb 5E5 directed...... to Tn-MUC1 was investigated using BiaCore. The availability of Tn-MUC1 on the surface of breast cancer cells was evaluated by immunohistochemistry, confocal microscopy, and flow cytometry, followed by in vitro assessment of antibody-dependent cellular cytotoxicity by mAb 5E5. Biacore analysis...... is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity suggesting that antibodies targeting glycopeptide epitopes on mucins are strong candidates for cancer-specific immunotherapies....

  1. Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies.

    Science.gov (United States)

    Maggi, Maristella; Scotti, Claudia

    2017-08-01

    Single domain antibodies (sdAbs) are small antigen-binding domains derived from naturally occurring, heavy chain-only immunoglobulins isolated from camelid and sharks. They maintain the same binding capability of full-length IgGs but with improved thermal stability and permeability, which justifies their scientific, medical and industrial interest. Several described recombinant forms of sdAbs have been produced in different hosts and with different strategies. Here we present an optimized method for a time-saving, high yield production and extraction of a poly-histidine-tagged sdAb from Escherichia coli classical inclusion bodies. Protein expression and extraction were attempted using 4 different methods (e.g. autoinducing or IPTG-induced soluble expression, non-classical and classical inclusion bodies). The best method resulted to be expression in classical inclusion bodies and urea-mediated protein extraction which yielded 60-70 mg/l bacterial culture. The method we here describe can be of general interest for an enhanced and efficient heterologous expression of sdAbs for research and industrial purposes. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Pairing Alpaca and Llama-Derived Single Domain Antibodies to Enhance Immunoassays for Ricin

    Directory of Open Access Journals (Sweden)

    Kendrick B. Turner

    2017-02-01

    Full Text Available Previously, our group isolated and evaluated anti-ricin single domain antibodies (sdAbs derived from llamas, engineered them to further increase their thermal stability, and utilized them for the development of sensitive immunoassays. In work focused on the development of therapeutics, Vance et al. 2013 described anti-ricin sdAbs derived from alpacas. Herein, we evaluated the utility of selected alpaca-derived anti-ricin sdAbs for detection applications, and engineered an alpaca-derived sdAb to increase its melting temperature, providing a highly thermal stable reagent for use in ricin detection. Four of the alpaca-derived anti-ricin A-chain sdAbs were produced and characterized. All four bound to epitopes that overlapped with our previously described llama sdAbs. One alpaca sdAb, F6, was found to possess both a high melting temperature (73 °C and to work optimally with a thermally stable llama anti-ricin sdAb in sandwich assays for ricin detection. We employed a combination of consensus sequence mutagenesis and the addition of a non-canonical disulfide bond to further enhance the thermal stability of F6 to 85 °C. It is advantageous to have a choice of recognition reagents when developing assays. This work resulted in defining an additional pair of highly thermal stable sdAbs for the sensitive detection of ricin.

  3. Antibody administration in experimental influenza increases survival and enhances the effect of oseltamivir

    DEFF Research Database (Denmark)

    Pourroy, Brit Naldahl Jessen; Kolmos, Hans Jørn; Nielsen, Lars Peter

    2012-01-01

    and treatment of a number of infectious diseases. In this experimental study anti-influenza antibodies were passively administrated to mice, subsequently they were infected with influenza virus and treated with oseltamivir. The aim was to investigate, if anti-influenza antibodies influenced the out come...... be considered in the in control of influenza....

  4. Antibody-dependent cellular inhibition is associated with reduced risk against febrile malaria in a longitudinal cohort study involving Ghanaian children

    DEFF Research Database (Denmark)

    Tiendrebeogo, Regis W; Adu, Bright; Singh, Susheel K

    2015-01-01

    The antibody-dependent respiratory burst and opsonic phagocytosis assays have been associated with protection against malaria; however, other mechanisms may also be involved. The antibody-dependent cellular inhibition (ADCI) assay is yet to be correlated with protection in longitudinal cohort stu...... of the ADCI assay as a correlate of protection to guide malaria vaccine studies.......The antibody-dependent respiratory burst and opsonic phagocytosis assays have been associated with protection against malaria; however, other mechanisms may also be involved. The antibody-dependent cellular inhibition (ADCI) assay is yet to be correlated with protection in longitudinal cohort...... studies (LCS). We investigated the relationship between ADCI activity of immunoglobulin G before malaria season and risk of malaria in a LCS involving Ghanaian children. High ADCI activity was significantly associated with reduced risk against malaria. Findings here suggest a potential usefulness...

  5. Dependence of enhanced asymmetry-induced transport on collision frequency

    International Nuclear Information System (INIS)

    Eggleston, D. L.

    2014-01-01

    A single-particle code with collisional effects is used to study how asymmetry-induced radial transport in a non-neutral plasma depends on collision frequency. For asymmetries of the form ϕ 1 (r) cos(kz) cos(ωt−lθ), two sources for the transport have been identified: resonant particles and axially trapped particles. The simulation shows that this latter type, which occurs near the radius where ω matches the azimuthal rotation frequency ω R , is usually dominant at low collision frequency ν but becomes negligible at higher ν. This behavior can be understood by noting that axially trapped particles have a lower trapping frequency than resonant particles. In the low ν (banana) regime, the radial oscillations have amplitude Δr ≈ v r /ω T , so axially trapped particles dominate, and the transport may even exceed the resonant particle plateau regime level. As ν increases, collisions start to interrupt the slower axially trapped particle oscillations, while the resonant particles are still in the banana regime, so the axially trapped particle contribution to the transport decreases. At the largest ν values, axially trapped particle transport is negligible and the observed diffusion coefficient matches that given by plateau regime resonant particle theory. Heuristic models based on these considerations give reasonable agreement with the observed scaling laws for the value of the collision frequency where axially trapped particle transport starts to decrease and for the enhancement of the diffusion coefficient produced by axially trapped particles

  6. RNA Sensors Enable Human Mast Cell Anti-Viral Chemokine Production and IFN-Mediated Protection in Response to Antibody-Enhanced Dengue Virus Infection

    Science.gov (United States)

    Huang, Yan Y.; Haidl, Ian D.; Al-Afif, Ayham; Marshall, Jean S.; Anderson, Robert

    2012-01-01

    Dengue hemorrhagic fever and/or dengue shock syndrome represent the most serious pathophysiological manifestations of human dengue virus infection. Despite intensive research, the mechanisms and important cellular players that contribute to dengue disease are unclear. Mast cells are tissue-resident innate immune cells that play a sentinel cell role in host protection against infectious agents via pathogen-recognition receptors by producing potent mediators that modulate inflammation, cell recruitment and normal vascular homeostasis. Most importantly, mast cells are susceptible to antibody-enhanced dengue virus infection and respond with selective cytokine and chemokine responses. In order to obtain a global view of dengue virus-induced gene regulation in mast cells, primary human cord blood-derived mast cells (CBMCs) and the KU812 and HMC-1 mast cell lines were infected with dengue virus in the presence of dengue-immune sera and their responses were evaluated at the mRNA and protein levels. Mast cells responded to antibody-enhanced dengue virus infection or polyinosiniċpolycytidylic acid treatment with the production of type I interferons and the rapid and potent production of chemokines including CCL4, CCL5 and CXCL10. Multiple interferon-stimulated genes were also upregulated as well as mRNA and protein for the RNA sensors PKR, RIG-I and MDA5. Dengue virus-induced chemokine production by KU812 cells was significantly modulated by siRNA knockdown of RIG-I and PKR, in a negative and positive manner, respectively. Pretreatment of fresh KU812 cells with supernatants from dengue virus-infected mast cells provided protection from subsequent infection with dengue virus in a type I interferon-dependent manner. These findings support a role for tissue-resident mast cells in the early detection of antibody-enhanced dengue virus infection via RNA sensors, the protection of neighbouring cells through interferon production and the potential recruitment of leukocytes via

  7. RNA sensors enable human mast cell anti-viral chemokine production and IFN-mediated protection in response to antibody-enhanced dengue virus infection.

    Directory of Open Access Journals (Sweden)

    Michael G Brown

    Full Text Available Dengue hemorrhagic fever and/or dengue shock syndrome represent the most serious pathophysiological manifestations of human dengue virus infection. Despite intensive research, the mechanisms and important cellular players that contribute to dengue disease are unclear. Mast cells are tissue-resident innate immune cells that play a sentinel cell role in host protection against infectious agents via pathogen-recognition receptors by producing potent mediators that modulate inflammation, cell recruitment and normal vascular homeostasis. Most importantly, mast cells are susceptible to antibody-enhanced dengue virus infection and respond with selective cytokine and chemokine responses. In order to obtain a global view of dengue virus-induced gene regulation in mast cells, primary human cord blood-derived mast cells (CBMCs and the KU812 and HMC-1 mast cell lines were infected with dengue virus in the presence of dengue-immune sera and their responses were evaluated at the mRNA and protein levels. Mast cells responded to antibody-enhanced dengue virus infection or polyinosiniċpolycytidylic acid treatment with the production of type I interferons and the rapid and potent production of chemokines including CCL4, CCL5 and CXCL10. Multiple interferon-stimulated genes were also upregulated as well as mRNA and protein for the RNA sensors PKR, RIG-I and MDA5. Dengue virus-induced chemokine production by KU812 cells was significantly modulated by siRNA knockdown of RIG-I and PKR, in a negative and positive manner, respectively. Pretreatment of fresh KU812 cells with supernatants from dengue virus-infected mast cells provided protection from subsequent infection with dengue virus in a type I interferon-dependent manner. These findings support a role for tissue-resident mast cells in the early detection of antibody-enhanced dengue virus infection via RNA sensors, the protection of neighbouring cells through interferon production and the potential recruitment of

  8. Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner.

    Science.gov (United States)

    Mullarkey, Caitlin E; Bailey, Mark J; Golubeva, Diana A; Tan, Gene S; Nachbagauer, Raffael; He, Wenqian; Novakowski, Kyle E; Bowdish, Dawn M; Miller, Matthew S; Palese, Peter

    2016-10-04

    Broadly neutralizing antibodies that recognize the conserved hemagglutinin (HA) stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR) interactions for optimal protection in vivo Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using an in vitro assay to detect the production of reactive oxygen species (ROS), we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR) engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection. The present study provides evidence that broadly neutralizing HA stalk-specific antibodies induce downstream Fc-mediated neutrophil effector functions. In addition to their ability to

  9. Immune antibodies and helminth products drive CXCR2-dependent macrophage-myofibroblast crosstalk to promote intestinal repair.

    Directory of Open Access Journals (Sweden)

    Julia Esser-von Bieren

    2015-03-01

    Full Text Available Helminth parasites can cause considerable damage when migrating through host tissues, thus making rapid tissue repair imperative to prevent bleeding and bacterial dissemination particularly during enteric infection. However, how protective type 2 responses targeted against these tissue-disruptive multicellular parasites might contribute to homeostatic wound healing in the intestine has remained unclear. Here, we observed that mice lacking antibodies (Aid-/- or activating Fc receptors (Fcrg-/- displayed impaired intestinal repair following infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb, whilst transfer of immune serum could partially restore chemokine production and rescue wound healing in Aid-/- mice. Impaired healing was associated with a reduced expression of CXCR2 ligands (CXCL2/3 by macrophages (MΦ and myofibroblasts (MF within intestinal lesions. Whilst antibodies and helminths together triggered CXCL2 production by MΦ in vitro via surface FcR engagement, chemokine secretion by intestinal MF was elicited by helminths directly via Fcrg-chain/dectin2 signaling. Blockade of CXCR2 during Hpb challenge infection reproduced the delayed wound repair observed in helminth infected Aid-/- and Fcrg-/- mice. Finally, conditioned media from human MΦ stimulated with infective larvae of the helminth Ascaris suum together with immune serum, promoted CXCR2-dependent scratch wound closure by human MF in vitro. Collectively our findings suggest that helminths and antibodies instruct a chemokine driven MΦ-MF crosstalk to promote intestinal repair, a capacity that may be harnessed in clinical settings of impaired wound healing.

  10. Passive immunization with a nicotine-specific monoclonal antibody decreases brain nicotine levels but does not precipitate withdrawal in nicotine-dependent rats

    OpenAIRE

    Roiko, Samuel A.; Harris, Andrew C.; LeSage, Mark G.; Keyler, Daniel E.; Pentel, Paul R.

    2009-01-01

    Vaccination against nicotine is under investigation as a treatment for tobacco dependence. Passive immunization with nicotine-specific antibodies represents a complementary strategy to vaccination. A potential adverse effect of passive immunization in nicotine-dependent individuals is that it may lead to a rapid reduction in brain nicotine levels and trigger withdrawal. The goal of this study was to determine if passive immunization with the nicotine-specific monoclonal antibody Nic311 precip...

  11. Development of a Fully Human Anti-PDGFRβ Antibody That Suppresses Growth of Human Tumor Xenografts and Enhances Antitumor Activity of an Anti-VEGFR2 Antibody

    Directory of Open Access Journals (Sweden)

    Juqun Shen

    2009-06-01

    Full Text Available Platelet-derived growth factor receptor β (PDGFRβ is upregulated in most of solid tumors. It is expressed by pericytes/smooth muscle cells, fibroblast, macrophage, and certain tumor cells. Several PDGF receptor-related antagonists are being developed as potential antitumor agents and have demonstrated promising antitumor activity in both preclinical and clinical settings. Here, we produced a fully human neutralizing antibody, IMC-2C5, directed against PDGFRβ from an antibody phage display library. IMC-2C5 binds to both human and mouse PDGFRβ and blocks PDGF-B from binding to the receptor. IMC-2C5 also blocks ligand-stimulated activation of PDGFRβ and downstream signaling molecules in tumor cells. In animal studies, IMC-2C5 significantly delayed the growth of OVCAR-8 and NCI-H460 human tumor xenografts in nude mice but failed to show antitumor activities in OVCAR-5 and Caki-1 xenografts. Our results indicate that the antitumor efficacy of IMC-2C5 is primarily due to its effects on tumor stroma, rather than on tumor cells directly. Combination of IMC-2C5 and DC101, an anti-mouse vascular endothelial growth factor receptor 2 antibody, resulted in significantly enhanced antitumor activity in BxPC-3, NCI-H460, and HCT-116 xenografts, compared with DC101 alone, and the trend of additive effects to DC101 treatment in several other tumor models. ELISA analysis of NCI-H460 tumor homogenates showed that IMC-2C5 attenuated protein level of vascular endothelial growth factor and basic fibroblast growth factor elevated by DC101 treatment. Finally, IMC-2C5 showed a trend of additive effects when combined with DC101/chemotherapy in MIA-PaCa-2 and NCI-H460 models. Taken together, these results lend great support to the use of PDGFRβ antagonists in combination with other antiangiogenic agents in the treatment of a broad range of human cancers.

  12. Trastuzumab mediates antibody-dependent cell-mediated cytotoxicity and phagocytosis to the same extent in both adjuvant and metastatic HER2/neu breast cancer patients.

    Science.gov (United States)

    Petricevic, Branka; Laengle, Johannes; Singer, Josef; Sachet, Monika; Fazekas, Judit; Steger, Guenther; Bartsch, Rupert; Jensen-Jarolim, Erika; Bergmann, Michael

    2013-12-12

    Monoclonal antibodies (mAb), such as trastuzumab are a valuable addition to breast cancer therapy. Data obtained from neoadjuvant settings revealed that antibody-dependent cell-mediated cytotoxicity (ADCC) is a major mechanism of action for the mAb trastuzumab. Conflicting results still call into question whether disease progression, prolonged treatment or concomitant chemotherapy influences ADCC and related immunological phenomena. We analyzed the activity of ADCC and antibody-dependent cell-mediated phagocytosis (ADCP) of peripheral blood mononuclear cells (PBMCs) from human epidermal growth factor receptor 2 (HER2/neu) positive breast cancer patients receiving trastuzumab therapy either in an adjuvant (n = 13) or metastatic (n = 15) setting as well as from trastuzumab treatment-naive (t-naive) HER2/neu negative patients (n = 15). PBMCs from healthy volunteers (n = 24) were used as controls. ADCC and ADCP activity was correlated with the expression of antibody binding Fc-gamma receptor (FcγR)I (CD64), FcγRII (CD32) and FcγRIII (CD16) on CD14+ (monocytes) and CD56+ (NK) cells, as well as the expression of CD107a+ (LAMP-1) on CD56+ cells and the total amount of CD4+CD25+FOXP3+ (Treg) cells. In metastatic patients, markers were correlated with progression-free survival (PFS). ADCC activity was significantly down regulated in metastatic, adjuvant and t-naive patient cohorts as compared to healthy controls. Reduced ADCC activity was inversely correlated with the expression of CD107a on CD56+ cells in adjuvant patients. ADCC and ADCP activity of the patient cohorts were similar, regardless of treatment duration or additional chemotherapy. PFS in metastatic patients inversely correlated with the number of peripheral Treg cells. The reduction of ADCC in patients as compared to healthy controls calls for adjuvant strategies, such as immune-enhancing agents, to improve the activity of trastuzumab. However, efficacy of trastuzumab-specific ADCC and ADCP appears not to

  13. A generic mechanism in Neisseria meningitidis for enhanced resistance against bactericidal antibodies

    OpenAIRE

    Uria, Maria Jose; Zhang, Qian; Li, Yanwen; Chan, Angel; Exley, Rachel M.; Gollan, Bridget; Chan, Hannah; Feavers, Ian; Yarwood, Andy; Abad, Raquel; Borrow, Ray; Fleck, Roland A.; Mulloy, Barbara; Vazquez, Julio A.; Tang, Christoph M.

    2008-01-01

    The presence of serum bactericidal antibodies is a proven correlate of protection against systemic infection with the important human pathogen Neisseria meningitidis. We have identified three serogroup C N. meningitidis (MenC) isolates recovered from patients with invasive meningococcal disease that resist killing by bactericidal antibodies induced by the MenC conjugate vaccine. None of the patients had received the vaccine, which has been successfully introduced in countries in North America...

  14. Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner

    Directory of Open Access Journals (Sweden)

    Caitlin E. Mullarkey

    2016-10-01

    Full Text Available Broadly neutralizing antibodies that recognize the conserved hemagglutinin (HA stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR interactions for optimal protection in vivo. Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using an in vitro assay to detect the production of reactive oxygen species (ROS, we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection.

  15. Mast cell activation is enhanced by Tim1:Tim4 interaction but not by Tim-1 antibodies [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Binh Phong

    2016-07-01

    Full Text Available Polymorphisms in the T cell (or transmembrane immunoglobulin and mucin domain 1 (TIM-1 gene, particularly in the mucin domain, have been associated with atopy and allergic diseases in mice and human. Genetic- and antibody-mediated studies revealed that Tim-1 functions as a positive regulator of Th2 responses, while certain antibodies to Tim-1 can exacerbate or reduce allergic lung inflammation. Tim-1 can also positively regulate the function of B cells, NKT cells, dendritic cells and mast cells. However, the precise molecular mechanisms by which Tim-1 modulates immune cell function are currently unknown. In this study, we have focused on defining Tim-1-mediated signaling pathways that enhance mast cell activation through the high affinity IgE receptor (FceRI. Using a Tim-1 mouse model lacking the mucin domain (Tim-1Dmucin, we show for the first time that the polymorphic Tim-1 mucin region is dispensable for normal mast cell activation. We further show that Tim-4 cross-linking of Tim-1 enhances select signaling pathways downstream of FceRI in mast cells, including mTOR-dependent signaling, leading to increased cytokine production but without affecting degranulation.

  16. Transcription-factor-dependent enhancer transcription defines a gene regulatory network for cardiac rhythm.

    Science.gov (United States)

    Yang, Xinan H; Nadadur, Rangarajan D; Hilvering, Catharina Re; Bianchi, Valerio; Werner, Michael; Mazurek, Stefan R; Gadek, Margaret; Shen, Kaitlyn M; Goldman, Joseph Aaron; Tyan, Leonid; Bekeny, Jenna; Hall, Johnathon M; Lee, Nutishia; Perez-Cervantes, Carlos; Burnicka-Turek, Ozanna; Poss, Kenneth D; Weber, Christopher R; de Laat, Wouter; Ruthenburg, Alexander J; Moskowitz, Ivan P

    2017-12-27

    The noncoding genome is pervasively transcribed. Noncoding RNAs (ncRNAs) generated from enhancers have been proposed as a general facet of enhancer function and some have been shown to be required for enhancer activity. Here we examine the transcription-factor-(TF)-dependence of ncRNA expression to define enhancers and enhancer-associated ncRNAs that are involved in a TF-dependent regulatory network. TBX5, a cardiac TF, regulates a network of cardiac channel genes to maintain cardiac rhythm. We deep sequenced wildtype and Tbx5 -mutant mouse atria, identifying ~2600 novel Tbx5 -dependent ncRNAs. Tbx5-dependent ncRNAs were enriched for tissue-specific marks of active enhancers genome-wide. Tbx5-dependent ncRNAs emanated from regions that are enriched for TBX5-binding and that demonstrated Tbx5-dependent enhancer activity. Tbx5 -dependent ncRNA transcription provided a quantitative metric of Tbx5 -dependent enhancer activity, correlating with target gene expression. We identified RACER , a novel Tbx5 -dependent long noncoding RNA (lncRNA) required for the expression of the calcium-handling gene Ryr2 . We illustrate that TF-dependent enhancer transcription can illuminate components of TF-dependent gene regulatory networks.

  17. Antibody-enhanced dengue disease generates a marked CNS inflammatory response in the black-tufted marmoset Callithrix penicillata.

    Science.gov (United States)

    Vasconcelos, Barbara Cristina Baldez; Vieira, Juliana Almeida; Silva, Geane Oliveira; Fernandes, Taiany Nogueira; Rocha, Luciano Chaves; Viana, André Pereira; Serique, Cássio Diego Sá; Filho, Carlos Santos; Bringel, Raissa Aires Ribeiro; Teixeira, Francisco Fernando Dacier Lobato; Ferreira, Milene Silveira; Casseb, Samir Mansour Moraes; Carvalho, Valéria Lima; de Melo, Karla Fabiane Lopes; de Castro, Paulo Henrique Gomes; Araújo, Sanderson Corrêa; Diniz, José Antonio Picanço; Demachki, Samia; Anaissi, Ana Karyssa Mendes; Sosthenes, Marcia Consentino Kronka; Vasconcelos, Pedro Fernando da Costa; Anthony, Daniel Clive; Diniz, Cristovam Wanderley Picanço; Diniz, Daniel Guerreiro

    2016-02-01

    Severe dengue disease is often associated with long-term neurological impairments, but it is unclear what mechanisms are associated with neurological sequelae. Previously, we demonstrated antibody-enhanced dengue disease (ADE) dengue in an immunocompetent mouse model with a dengue virus 2 (DENV2) antibody injection followed by DENV3 virus infection. Here we migrated this ADE model to Callithrix penicillata. To mimic human multiple infections of endemic zones where abundant vectors and multiple serotypes co-exist, three animals received weekly subcutaneous injections of DENV3 (genotype III)-infected supernatant of C6/36 cell cultures, followed 24 h later by anti-DENV2 antibody for 12 weeks. There were six control animals, two of which received weekly anti-DENV2 antibodies, and four further animals received no injections. After multiple infections, brain, liver, and spleen samples were collected and tissue was immunolabeled for DENV3 antigens, ionized calcium binding adapter molecule 1, Ki-67, TNFα. There were marked morphological changes in the microglial population of ADE monkeys characterized by more highly ramified microglial processes, higher numbers of trees and larger surface areas. These changes were associated with intense TNFα-positive immunolabeling. It is unclear why ADE should generate such microglial activation given that IgG does not cross the blood-brain barrier, but this study reveals that in ADE dengue therapy targeting the CNS host response is likely to be important. © 2015 Japanese Society of Neuropathology.

  18. Conserved epitope on several human vitamin K-dependent proteins: location of the antigenic site and influence of metal ions on antibody binding

    International Nuclear Information System (INIS)

    Church, W.R.; Messier, T.; Howard, P.R.; Amiral, J.; Meyer, D.; Mann, K.G.

    1988-01-01

    A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of 125 I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 x 10 -8 to 1 x 10 -6 M. Chemical treatment of prothrombin with a variety of agents did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. Increasing concentrations of Ca 2+ , Mg 2+ , or Mn 2+ partially inhibited binding of 125 I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively. The antigenic site thus recognized by monoclonal antibody H-11 is located at the amino-terminal region in the highly conserved γ-carboxyglutamic acid-containing domains of several, but not all, vitamin K-dependent proteins

  19. B7h-expressing dendritic cells and plasma B cells mediate distinct outcomes of ICOS costimulation in T cell-dependent antibody responses

    Directory of Open Access Journals (Sweden)

    Larimore Kevin

    2012-06-01

    Full Text Available Abstract Background The ICOS-B7h costimulatory receptor-ligand pair is required for germinal center formation, the production of isotype-switched antibodies, and antibody affinity maturation in response to T cell-dependent antigens. However, the potentially distinct roles of regulated B7h expression on B cells and dendritic cells in T cell-dependent antibody responses have not been defined. Results We generated transgenic mice with lineage-restricted B7h expression to assess the cell-type specific roles of B7h expression on B cells and dendritic cells in regulating T cell-dependent antibody responses. Our results show that endogenous B7h expression is reduced on B cells after activation in vitro and is also reduced in vivo on antibody-secreting plasma B cells in comparison to both naïve and germinal center B cells from which they are derived. Increasing the level of B7h expression on activated and plasma B cells in B-B7hTg mice led to an increase in the number of antibody-secreting plasma cells generated after immunization and a corresponding increase in the concentration of antigen-specific high affinity serum IgG antibodies of all isotypes, without affecting the number of responding germinal center B cells. In contrast, ICOS costimulation mediated by dendritic cells in DC-B7hTg mice contributed to germinal center formation and selectively increased IgG2a production without affecting the overall magnitude of antibody responses. Conclusions Using transgenic mice with lineage-restricted B7h expression, we have revealed distinct roles of ICOS costimulation mediated by dendritic cells and B cells in the regulation of T cell-dependent antibody responses.

  20. Mannosylated mucin-type immunoglobulin fusion proteins enhance antigen-specific antibody and T lymphocyte responses.

    Directory of Open Access Journals (Sweden)

    Gustaf Ahlén

    Full Text Available Targeting antigens to antigen-presenting cells (APC improve their immunogenicity and capacity to induce Th1 responses and cytotoxic T lymphocytes (CTL. We have generated a mucin-type immunoglobulin fusion protein (PSGL-1/mIgG(2b, which upon expression in the yeast Pichia pastoris became multivalently substituted with O-linked oligomannose structures and bound the macrophage mannose receptor (MMR and dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN with high affinity in vitro. Here, its effects on the humoral and cellular anti-ovalbumin (OVA responses in C57BL/6 mice are presented.OVA antibody class and subclass responses were determined by ELISA, the generation of anti-OVA CTLs was assessed in (51Cr release assays using in vitro-stimulated immune spleen cells from the different groups of mice as effector cells and OVA peptide-fed RMA-S cells as targets, and evaluation of the type of Th cell response was done by IFN-γ, IL-2, IL-4 and IL-5 ELISpot assays.Immunizations with the OVA - mannosylated PSGL-1/mIgG(2b conjugate, especially when combined with the AbISCO®-100 adjuvant, lead to faster, stronger and broader (with regard to IgG subclass OVA IgG responses, a stronger OVA-specific CTL response and stronger Th1 and Th2 responses than if OVA was used alone or together with AbISCO®-100. Also non-covalent mixing of mannosylated PSGL-1/mIgG(2b, OVA and AbISCO®-100 lead to relatively stronger humoral and cellular responses. The O-glycan oligomannoses were necessary because PSGL-1/mIgG(2b with mono- and disialyl core 1 structures did not have this effect.Mannosylated mucin-type fusion proteins can be used as versatile APC-targeting molecules for vaccines and as such enhance both humoral and cellular immune responses.

  1. The Amaranthus leucocarpus lectin enhances the anti-CD3 antibody-mediated activation of human peripheral blood CD4+ T cells.

    Science.gov (United States)

    Urrea, Francisco; Ortiz-Quintero, Blanca; Sanchez-Garcia, Francisco Javier; Blanco-Favela, Francisco; Garfias, Yonathan; Lascurain, Ricardo; Zenteno, Edgar

    2010-08-01

    Activation of CD4(+) T cells plays a main role in adaptive immune response by regulating cellular and humoral immunity via processes associated with changes in cell surface oligosaccharide receptors. Lectins are glycoproteins that specifically recognize oligosaccharides and have been used to characterize changes in oligosaccharides present on T cell surface and their effects on activation. A lectin from Amaranthus leucocarpus seeds (ALL) is specific for glycoprotein structures containing galactose-N-acetylgalactosamine and is able to bind to human and murine CD4(+) T cells, however, its effect on activation remains unclear. We examined the effect of ALL on the activation of peripheral blood human CD4(+) T cells and analyzed cell proliferation, expression of the activation-associated molecule CD25, secretion of the activation-dependent cytokine interleukin (IL)-2 and intracellular calcium influx changes using flow cytometry. CD4(+) T cells were stimulated with anti-CD3 antibodies that provided the first activation signal in the presence or absence of ALL. ALL alone did not induce CD4(+) T cell activation but when also stimulated with anti-CD3 antibodies, ALL up-regulated CD25 expression, cell proliferation, IL-2 secretion and an intracellular calcium influx in a dose-dependent manner. In addition, ALL recognized CD4(+) T cells expressing the CD69 and Ki67 molecules expressed only by activated T cells and induced production of the TH1-type cytokine interferon-gamma. Our findings indicate that ALL binds to human activated CD4(+) T cells and enhances the degree of activation of CD4(+) T cells that are stimulated with anti-CD3 antibodies. ALL provides a new tool for analyzing T cell activation mechanisms.

  2. Dependence of metal-enhanced fluorescence on surface roughness

    Science.gov (United States)

    François, Alexandre; Sciacca, Beniamino; Zuber, Agnieszka; Klantsataya, Elizaveta; Monro, Tanya M.

    2014-03-01

    Metal Enhanced Fluorescence (MEF) takes advantage of the coupling between surface plasmons, in either a metallic thin film or metallic nanoparticles, and fluorophores located in proximity of the metal, yielding an increase of the fluorophore emission. While MEF has been widely studied on metallic nanoparticles with the emphasis on creating brighter fluorescent labels, planar surfaces have not benefitted from the same attention. Here we investigate the influence of the surface roughness of a thin metallic film on the fluorescence enhancement. 50nm thick silver films were deposited on glass slides using either thermal evaporation with different evaporation currents or an electroless plating method based on the Tollens reaction to vary the surface roughness. Multiple layers of positively and negatively charged polyelectrolytes were deposited on top of the metallic coating to map out the enhancement factor as function of the gap between the metallic coating and fluorophore molecules covalently bound to the last polyelectrolyte layer. We show that fluorescence is enhanced by the presence of the metallic film, and in particular that the enhancement increases by a factor 3 to 40 for roughness ranging from 3 nm to 8 nm. Although these enhancement factors are modest compared to the enhancement produced by complex metallic nanoparticles or nano-patterned metallic thin films, the thin films used here are capable of supporting a plasmonic wave and offer the possibility of combining different techniques, such as surface plasmon resonance (with its higher refractive index sensitivity compared to localized plasmons) and MEF within a single device.

  3. Ah receptor mediated suppression of the antibody response in mice is primarily dependent on the Ah phenotype of lymphoid tissue

    International Nuclear Information System (INIS)

    Silkworth, J.B.; Antrim, L.A.; Sack, G.

    1986-01-01

    Halogenated aromatic hydrocarbons act through the aromatic hydrocarbon (Ah) receptor in mice to produce a series of toxic effects of the immune system. The receptor protein is a product of the Ah gene locus. Ah responsive (Ahb/Ahb) mice express a high affinity receptor in both lymphoid and nonlymphoid tissues whereas nonresponsive Ahd/Ahd mice express a poor affinity receptor. To determine the role of the Ah receptor of lymphoid tissue relative to that of nonlymphoid tissue in the induction of immune impairment, bone marrow was used to reconstitute lethally irradiated mice of the same or opposite Ah phenotype. All mice were given 3,3',4,4'-tetrachlorobiphenyl (35 and 350 mumol/kg) ip 2 days before immunization with sheep erythrocytes (SRBC). The immune response to this T dependent antigen and organ weights were determined 5 or 7 days later in normal or chimeric mice, respectively. Monoclonal Lyt 1.1 and Lyt 1.2 antibodies were used to establish the origin of the cells which repopulated the chimeric thymuses. The immune responses of both BALB/cBy (Ahb/Ahb) and the BALB/cBy X DBA/2 hybrid, CByD2F1 (Ahb/Ahd), were significantly suppressed but DBA/2 mice were unaffected. The immune responses of chimeric BALB/cBy----BALB/cBy and BALB/cBy----DBA/2 (donor----recipient) mice were also significantly suppressed and thymic atrophy was observed in both cases. The serum anti-SRBC antibody titers of DBA/2----BALB/cBy chimeras were also significantly decreased although not to the same extent as in BALB/cBy----DBA/2 mice. Chimeric DBA/2----DBA/2 mice were not affected. These results indicate that the sensitivity to Ah receptor mediated suppression of the antibody response is primarily determined by the Ah phenotype of the lymphoid tissue

  4. Instrumental learning: An animal model for sleep dependent memory enhancement

    NARCIS (Netherlands)

    Leenaars, Cathalijn H. C.; Girardi, Carlos E. N.; Joosten, Ruud N. J. M. A.; Lako, Irene M.; Ruimschotel, Emma; Hanegraaf, Maaike A. J.; Dematteis, Maurice; Feenstra, Matthijs G. P.; van Someren, Eus J. W.

    2013-01-01

    The relationship between learning and sleep is multifaceted; learning influences subsequent sleep characteristics, which may in turn influence subsequent memory. Studies in humans indicate that sleep may not only prevent degradation of acquired memories, but even enhance performance without further

  5. Vaccine and Monoclonal Antibody That Enhance Mouse Resistance to Candidiasis ▿

    Science.gov (United States)

    Xin, Hong; Cutler, Jim E.

    2011-01-01

    Previously we showed that antibodies specific for the glycan β-1,2-mannotriose [β-(Man)3] on the cell surface of Candida albicans protect mice against disseminated candidiasis (H. Xin, S. Dziadek, D. R. Bundle, and J. E. Cutler, Proc. Natl. Acad. Sci. U. S. A. 105:13526–13531, 2008). Furthermore, six 14-mer peptides that are within the N-terminal portion of C. albicans wall proteins were conjugated to the glycan in an attempt to create immunogenic glycopeptide conjugates. By a dendritic cell (DC)-based immunization approach, all were immunogenic and three of the six conjugates induced a high degree of protection in mice. Interestingly, whereas all six peptides induced antibody responses when used alone to pulse DCs for subsequent immunizations, three peptides induced protection, and one in particular, peptide Fba (derived from fructose-bisphosphate aldolase), induced robust protective responses and is the focus of the current work. Fba peptide is not restricted by the major histocompatibility complex class II (MHC-II), as it induced anti-Fba antibodies in mice of different H-2 haplotypes and in rabbits. Furthermore, the peptide induced protection against disease caused by different C. albicans strains. Partial protection was achieved when alum was used in place of DCs for Fba immunizations. The passive transfer of immune sera from Fba-vaccinated mice, but not immune serum preabsorbed with fungal cells, conferred protection in naïve mice. This result, along with our finding that a monoclonal antibody specific for the peptide, E2-9 (IgM), protected mice against candidiasis, provide strong evidence that antibodies contribute to protection. Our work demonstrates the utility of cell wall peptides alone or as glycopeptides in vaccines designed for the induction of immunity against candidiasis and monoclonal antibodies as a rapid immunoprotective approach against the disease. PMID:21832099

  6. Clinical Cancer Therapy by NK Cells via Antibody-Dependent Cell-Mediated Cytotoxicity

    Directory of Open Access Journals (Sweden)

    Kory L. Alderson

    2011-01-01

    Full Text Available Natural killer (NK cells are powerful effector cells that can be directed to eliminate tumor cells through tumor-targeted monoclonal antibodies (mAbs. Some tumor-targeted mAbs have been successfully applied in the clinic and are included in the standard of care for certain malignancies. Strategies to augment the antitumor response by NK cells have led to an increased understanding of how to improve their effector responses. Next-generation reagents, such as molecularly modified mAbs and mAb-cytokine fusion proteins (immunocytokines, ICs designed to augment NK-mediated killing, are showing promise in preclinical and some clinical settings. Continued research into the antitumor effects induced by NK cells and tumor-targeted mAbs suggests that additional intrinsic and extrinsic factors may influence the antitumor response. Therefore more research is needed that focuses on evaluating which NK cell and tumor criteria are best predictive of a clinical response and which combination immunotherapy regimens to pursue for distinct clinical settings.

  7. Age-dependent prevalence of anti-Brucella antibodies in hooded seals Cystophora cristata.

    Science.gov (United States)

    Nymo, Ingebjørg H; Tryland, Morten; Frie, Anne Kirstine; Haug, Tore; Foster, Geoffrey; Rødven, Rolf; Godfroid, Jacques

    2013-11-06

    Investigations of hooded seals Cystophora cristata have revealed high prevalences of Brucella-positive seals in the reduced Northeast Atlantic stock, compared to the increasing Northwest Atlantic stock. This study evaluated the relation between Brucella-serostatus in seals in the Northeast Atlantic stock and age, sex, body condition and reproduction. Bacteriology documented which animals and organs were B. pinnipedialis positive. No relationship was observed between Brucella-serostatus and body condition or reproductive traits. Pups (seals >1 yr old, the mean probability of being seropositive decreased with age, with no seropositives older than 5 yr, indicating loss of antibody titre with either chronicity or clearance of infection. The latter explanation seems to be most likely as B. pinnipedialis has never been isolated from a hooded seal >18 mo old, which is consistent with findings in this study; B. pinnipedialis was isolated from the retropharyngeal lymph node in 1 seropositive yearling (1/21, 5%). We hypothesize that this serological and bacteriological pattern is due to environmental exposure to B. pinnipedialis early in life, with a subsequent clearance of infection. This raises the question of a reservoir of B. pinnipedialis in the hooded seal food web.

  8. Enhanced expression of a calcium-dependent protein kinase from ...

    Indian Academy of Sciences (India)

    Unknown

    Calcium-dependent protein kinase; degenerate primer; Funaria hygrometrica; nutrient; polymerase chain reaction; starvation ... Among the downstream targets of calcium in plants, calcium-dependent protein kinases (CDPKs) form an inter- esting class of ..... Saunders M J and Hepler P K 1983 Calcium antagonists and.

  9. Natural killer cell cytotoxicity and antibody-dependent cellular cytotoxicity to herpes simplex virus-infected cells in human pregnancy.

    Science.gov (United States)

    Gonik, B; Loo, L S; West, S; Kohl, S

    1987-01-01

    Natural killer cell (NKC) cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) represent the ability of human leukocyte effector cells to destroy target cells in the absence and presence of antibody, respectively. Since these immune systems play a pivotal role in the body's primary lines of defense against a variety of pathogens including herpes simplex virus (HSV), a study was undertaken to evaluate the influence of pregnancy on these systems. Eleven uncomplicated gravidas were followed serially through each trimester and compared to 11 nonpregnant female controls. Mononuclear cells were acquired by Ficoll-Hypaque centrifugation of heparinized blood. Chang liver cells infected with HSV-I were utilized as target cells in a 51Cr release assay. Mean NKC values in the pregnant patients were uniformly lower than in the controls. No similar decreases in ADCC activity were observed in a comparison between the two study populations. These data support previous observations suggesting that pregnancy represents a relatively immunocompromised state. Differences apparently exist between NKC and ADCC effector cell populations with regard to the influence of pregnancy. Although these physiologic alterations in immunoregulation may help support the fetoplacental allograph, detrimental conditions may exist regarding susceptibility to various pathogens such as HSV.

  10. Structural basis of activation-dependent binding of ligand-mimetic antibody AL-57 to integrin LFA-1

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Hongmin; Liu, Jin-huan; Yang, Wei; Springer, Timothy; Shimaoka, Motomu; Wang, Jia-huai; (CH-Boston); (DFCI)

    2010-09-21

    The activity of integrin LFA-1 ({alpha}{sub L}{beta}{sub 2}) to its ligand ICAM-1 is regulated through the conformational changes of its ligand-binding domain, the I domain of {alpha}{sub L} chain, from an inactive, low-affinity closed form (LA), to an intermediate-affinity form (IA), and then finally, to a high-affinity open form (HA). A ligand-mimetic human monoclonal antibody AL-57 (activated LFA-1 clone 57) was identified by phage display to specifically recognize the affinity-upregulated I domain. Here, we describe the crystal structures of the Fab fragment of AL-57 in complex with IA, as well as in its unligated form. We discuss the structural features conferring AL-57's strong selectivity for the high affinity, open conformation of the I domain. The AL-57-binding site overlaps the ICAM-1 binding site on the I domain. Furthermore, an antibody Asp mimics an ICAM Glu by forming a coordination to the metal-ion dependent adhesion site (MIDAS). The structure also reveals better shape complementarity and a more hydrophobic interacting interface in AL-57 binding than in ICAM-1 binding. The results explain AL-57's antagonistic mimicry of LFA-1's natural ligands, the ICAM molecules.

  11. Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2 Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

    Directory of Open Access Journals (Sweden)

    Masaki Kurogochi

    Full Text Available Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain, and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC and complement dependent cytotoxicity (CDC. To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases, one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2, high-mannose type (Man4-9GlcNAc2, and complex type (Man3GlcNAc3-4 N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL, the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1 were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q, and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2 was performed using SKBR-3 and BT-474 as target

  12. A bacterial signal peptidase enhances processing of a recombinant single chain antibody fragment in insect cells

    NARCIS (Netherlands)

    Ailor, E; Pathmanathan, J; Jongbloed, JDH; Betenbaugh, MJ

    1999-01-01

    The production of an antibody single chain fragment (scFv) in insect cells was accompanied by the formation of an insoluble intracellular precursor even with the inclusion of the bee melittin signal peptide. The presence of the precursor polypeptide suggests a limitation in the processing of the

  13. Modulation of immune response by bacterial lipopolysaccharide (LPS): cellular basis of stimulatory and inhibitory effects of LPS on the in vitro IGM antibody response to a T-dependent antigen

    International Nuclear Information System (INIS)

    Uchiyama, T.; Jacobs, D.M.

    1978-01-01

    The role of thymus-derived lymphocytes (T cells) in LPS modulation of T cell-development antibody responses has been investigated. We have assessed the effect of LPS on the primary anti-TNP response to TNP-SRBC of cultures of whole spleen cells or T cell-depleted spleen cells that were supplemented with various subpopulations of carrier-primed (SRBC) spleen cells. The TNP-PFC response was enhanced in the presence of irradiated SRBC-primed spleen cells by addition of 0.16 to 20 μg/ml LPS, but inhibition was observed when irradiation of primed cells was omitted. Enhancement but no inhibition occurred when added primed cells were first passed through a nylon wool column. LPS-mediated enhancement was dependent on a T cell in the primed population. These results suggest that LPS modulation of antibody synthesis is dependent on two populations of antigen-specific cells that have opposing effects on B cell responses to a T-dependent antigen: a helper cell that is irradiation resistant, nonadherent to nylon wool, and sensitive to anti-T cell serum, and a suppressor cell that is irradiation sensitive and adherent to nylon wool

  14. Plasmon enhanced fluoro-immunoassay using egg yolk antibodies for ultra-sensitive detection of herbicide diuron.

    Science.gov (United States)

    Sharma, Priyanka; Kukkar, Manil; Ganguli, Ashok K; Bhasin, Aman; Suri, C Raman

    2013-08-07

    Plasmon enhanced fluorescence immunoassay (PEFI) format has been reported in developing a sensitive heterogeneous fluoroimmunoassay for monitoring the phenylurea herbicide diuron. Computer-assisted molecular modeling was carried out to study the conformational and electrostatic effects of synthesized hapten for producing highly specific egg yolk antibody against a phenyl urea herbicide diuron. The generated antibodies were labeled with fluorescein isothiocyanate at different molar ratios and used as tracer in the developed fluorescence based immunoassay. The sensitivity of the assay format was enhanced by using silver nanoparticles tagged with bovine serum albumin as a new blocking reagent in the developed PEFI format. Enhancer treatment on the developed immunoassay showed a significant improvement of fluorescence signal intensity with approximately 10 fold increase in assay sensitivity. The immunoassay has a detection limit of 0.01 ng mL(-1) with good signal precision (~2%) in the optimum working concentration range between 1 pg mL(-1) to 10 μg mL(-1) of diuron. These findings facilitate high throughput fluorescence-based processes that could be useful in biology, drug discovery and compound screening applications.

  15. The ethnic distribution of antibodies to glutamic acid decarboxylase: presence and levels of insulin-dependent diabetes mellitus in Europid and Asian subjects.

    Science.gov (United States)

    Zimmet, P Z; Rowley, M J; Mackay, I R; Knowles, W J; Chen, Q Y; Chapman, L H; Serjeantson, S W

    1993-01-01

    Our objective was to ascertain the frequency of antibodies to glutamic acid decarboxylase (GAD) in Europids and four Asian ethnic groups with insulin-dependent diabetes mellitus (IDDM) to gain insight into why the prevalence and incidence of IDDM varies so widely among ethnic and/or geographically diverse population groups. The subjects in this study were Europid (n = 49), Japanese (n = 16), Thai (n = 7), Korean (n = 21), and Chinese (n = 13) persons with IDDM with a duration ranging from 5 to 14 years. There were similar numbers of healthy controls matched for each ethnic group. A validated radioimmunoprecipitation assay used GAD from pig brain radiolabeled with 125I using chloramine T. Islet cell cytoplasmic antibodies measured by indirect immunofluorescence were expressed as Juvenile Diabetes Foundation units. The prevalence of antibodies to GAD, compared with Europids (63%), was much lower in all Asian populations with IDDM: Japanese (31%), Thai (29%), Korean (5%), and Chinese (27%). The mean level of antibodies to GAD, however, among diabetics from each population who gave a positive reaction, was similar. For all groups, the prevalence of antibodies to GAD was much higher than that of islet cell cytoplasmic antibodies. Almost all IDDM subjects positive for islet cell antibodies had antibodies to GAD, but the converse did not hold. A radioimmunoprecipitation assay for antibodies to GAD applied to serum from subjects with IDDM in various ethnic groups showed that Europids with IDDM had a much higher prevalence of such antibodies than did Asians. This held for all ethnic groups, and particularly Koreans. Thus, among different populations, there may be etiologic heterogeneity of IDDM.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Data on enhanced expression and purification of camelid single domain antibodies from Escherichia coli classical inclusion bodies.

    Science.gov (United States)

    Maggi, Maristella; Scotti, Claudia

    2017-06-01

    Heterologous expression of high amounts of recombinant proteins is a milestone for research and industrial purposes. Single domain antibodies (sdAbs) are heavy-chain only antibody fragments with applications in the biotechnological, medical and industrial fields. The simple nature and small size of sdAbs allows for efficient expression of the soluble molecule in different hosts. However, in some cases, it results in low functional protein yield. To overcome this limitation, expression of a 6xHistag sdAb was attempted in different conditions in Escherichia coli BL21(DE3) cells. Data showed that high amount of sdAb can be expressed in E. coli classical inclusion bodies, efficiently extracted by urea in a short-time, and properly purified by metal ion affinity chromatography. These data originate from the research article "Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies" Maggi and Scotti (2017) [1] (DOI: http://dx.doi.org/10.1016/j.pep.2017.02.007).

  17. Increased half-life and enhanced potency of Fc-modified human PCSK9 monoclonal antibodies in primates.

    Directory of Open Access Journals (Sweden)

    Yijun Shen

    Full Text Available Blocking proprotein convertase subtilisin kexin type 9 (PCSK9 binding to low-density lipoprotein receptor (LDLR can profoundly lower plasma LDL levels. Two anti-PCKS9 monoclonal antibodies (mAbs, alirocumab and evolocumab, were approved by the FDA in 2015. The recommended dose is 75 mg to 150 mg every two weeks for alirocumab and 140mg every two weeks or 420 mg once a month for evolocumab. This study attempted to improve the pharmacokinetic properties of F0016A, an IgG1 anti-PCKS9 mAb, to generate biologically superior molecules. We engineered several variants with two or three amino acid substitutions in the Fc fragment based on prior knowledge. The Fc-modified mAbs exhibited increased binding to FcRn, resulting in prolonged serum half-life and enhanced efficacy in vivo. These results demonstrate that Fc-modified anti-PCKS9 antibodies may enable less frequent or lower dosing of antibodies by improved recycling into the blood.

  18. Ab-origin: an enhanced tool to identify the sourcing gene segments in germline for rearranged antibodies

    Directory of Open Access Journals (Sweden)

    Sun Jing

    2008-12-01

    Full Text Available Abstract Background In the adaptive immune system, variable regions of immunoglobulin (IG are encoded by random recombination of variable (V, diversity (D, and joining (J gene segments in the germline. Partitioning the functional antibody sequences to their sourcing germline gene segments is vital not only for understanding antibody maturation but also for promoting the potential engineering of the therapeutic antibodies. To date, several tools have been developed to perform such "trace-back" calculations. Yet, the predicting ability and processing volume of those tools vary significantly for different sets of data. Moreover, none of them give a confidence for immunoglobulin heavy diversity (IGHD identification. Developing fast, efficient and enhanced tools is always needed with the booming of immunological data. Results Here, a program named Ab-origin is presented. It is designed by batch query against germline databases based on empirical knowledge, optimized scoring scheme and appropriate parameters. Special efforts have been paid to improve the identification accuracy of the short and volatile region, IGHD. In particular, a threshold score for certain sensitivity and specificity is provided to give the confidence level of the IGHD identification. Conclusion When evaluated using different sets of both simulated data and experimental data, Ab-origin outperformed all the other five popular tools in terms of prediction accuracy. The features of batch query and confidence indication of IGHD identification would provide extra help to users. The program is freely available at http://mpsq.biosino.org/ab-origin/supplementary.html.

  19. Stability of a viral infection model with state-dependent delay, CTL and antibody immune responses

    Czech Academy of Sciences Publication Activity Database

    Rezunenko, Oleksandr

    2017-01-01

    Roč. 22, č. 4 (2017), s. 1547-1563 ISSN 1531-3492 R&D Projects: GA ČR(CZ) GA16-06678S Institutional support: RVO:67985556 Keywords : Evolution equations * Lyapunov stability * state-dependent delay * virus infection model Subject RIV: BC - Control Systems Theory OBOR OECD: Computer sciences, information science, bioinformathics (hardware development to be 2.2, social aspect to be 5.8) Impact factor: 0.994, year: 2016 http://library.utia.cas.cz/separaty/2017/AS/rezunenko-0476128.pdf

  20. Enhanced expression of a calcium-dependent protein kinase from ...

    Indian Academy of Sciences (India)

    Among the downstream targets of calcium in plants, calcium-dependent protein kinases (CDPKs) form an interesting class of kinases which are activated by calcium binding. They have been implicated in a diverse array of responses to hormonal and environmental stimuli. In order to dissect the role of CDPKs in the moss ...

  1. Enhanced expression of a calcium-dependent protein kinase

    Indian Academy of Sciences (India)

    Among the downstream targets of calcium in plants, calcium-dependent protein kinases (CDPKs) form an interesting class of kinases which are activated by calcium binding. They have been implicated in a diverse array of responses to hormonal and environmental stimuli. In order to dissect the role of CDPKs in the moss ...

  2. Molecular mechanisms of dengue virus infection : cell tropism, antibody-dependent enhancement, and cytokines

    NARCIS (Netherlands)

    Flipse, Jacobus

    2015-01-01

    Dengue is the most prevalent mosquito-borne viral disease in humans. Although most infections occur in the (sub)tropical areas, recent outbreaks in Italy and Madeira indicate that the virus is spreading into Europe. Despite its relevance, no vaccine or medications are available against this virus.

  3. Molecular mechanisms involved in antibody-dependent enhancement of dengue virus infection in humans

    NARCIS (Netherlands)

    Flipse, Jacky; Wilschut, Jan; Smit, Jolanda M.

    Dengue is the most common arthropod-borne viral infection in humans with similar to 50 million cases annually worldwide. In recent decades, a steady increase in the number of severe dengue cases has been seen. Severe dengue disease is most often observed in individuals that have pre-existing

  4. Transcription-factor-dependent enhancer transcription defines a gene regulatory network for cardiac rhythm

    NARCIS (Netherlands)

    Yang, Xinan H; Nadadur, Rangarajan D; Hilvering, Catharina Re; Bianchi, Valerio; Werner, Michael; Mazurek, Stefan R; Gadek, Margaret; Shen, Kaitlyn M; Goldman, Joseph Aaron; Tyan, Leonid; Bekeny, Jenna; Hall, Johnathan M; Lee, Nutishia; Perez-Cervantes, Carlos; Burnicka-Turek, Ozanna; Poss, Kenneth D; Weber, Christopher R; de Laat, Wouter; Ruthenburg, Alexander J; Moskowitz, Ivan P

    2017-01-01

    The noncoding genome is pervasively transcribed. Noncoding RNAs (ncRNAs) generated from enhancers have been proposed as a general facet of enhancer function and some have been shown to be required for enhancer activity. Here we examine the transcription-factor-(TF)-dependence of ncRNA expression to

  5. Concentration Dependence of Gold Nanoparticles for Fluorescence Enhancement

    Science.gov (United States)

    Solomon, Joel; Wittmershaus, Bruce

    Noble metal nanoparticles possess a unique property known as surface plasmon resonance in which the conduction electrons oscillate due to incoming light, dramatically increasing their absorption and scattering of light. The oscillating electrons create a varying electric field that can affect nearby molecules. The fluorescence and photostability of fluorophores can be enhanced significantly when they are near plasmonic nanoparticles. This effect is called metal enhanced fluorescence (MEF). MEF from two fluorescence organic dyes, Lucifer Yellow CH and Riboflavin, was measured with different concentrations of 50-nm colloidal gold nanoparticles (Au-NP). The concentration range of Au-NP was varied from 2.5 to 250 pM. To maximize the interaction, the dyes were chosen so their emission spectra had considerable overlap with the absorption spectra of the Au-NP, which is common in MEF studies. If the dye molecules are too close to the surface of Au-NP, fluorescence quenching can occur instead of MEF. To try to observe this difference, silica-coated Au-NP were compared to citrate-based Au-NP; however, fluorescence quenching was observed with both Au-NP. This material is based upon work supported by the National Science Foundation under Grant Number NSF-ECCS-1306157.

  6. Sleep enhances false memories depending on general memory performance.

    Science.gov (United States)

    Diekelmann, Susanne; Born, Jan; Wagner, Ullrich

    2010-04-02

    Memory is subject to dynamic changes, sometimes giving rise to the formation of false memories due to biased processes of consolidation or retrieval. Sleep is known to benefit memory consolidation through an active reorganization of representations whereas acute sleep deprivation impairs retrieval functions. Here, we investigated whether sleep after learning and sleep deprivation at retrieval enhance the generation of false memories in a free recall test. According to the Deese, Roediger, McDermott (DRM) false memory paradigm, subjects learned lists of semantically associated words (e.g., "night", "dark", "coal", etc.), lacking the strongest common associate or theme word (here: "black"). Free recall was tested after 9h following a night of sleep, a night of wakefulness (sleep deprivation) or daytime wakefulness. Compared with memory performance after a retention period of daytime wakefulness, both post-learning nocturnal sleep as well as acute sleep deprivation at retrieval significantly enhanced false recall of theme words. However, these effects were only observed in subjects with low general memory performance. These data point to two different ways in which sleep affects false memory generation through semantic generalization: one acts during consolidation on the memory trace per se, presumably by active reorganization of the trace in the post-learning sleep period. The other is related to the recovery function of sleep and affects cognitive control processes of retrieval. Both effects are unmasked when the material is relatively weakly encoded. Crown Copyright 2009. Published by Elsevier B.V. All rights reserved.

  7. Attention enhancing effects of methylphenidate are age-dependent.

    Science.gov (United States)

    Bhattacharya, Shevon E; Shumsky, Jed S; Waterhouse, Barry D

    2015-01-01

    The psychostimulant methylphenidate (MPH, Ritalin®) is used to treat a variety of cognitive disorders. MPH is also popular among healthy individuals, including the elderly, for its ability to focus attention and improve concentration, but these effects have not been shown to be comparable between aged and adult subjects. Thus, we tested whether MPH would improve performance in sustained attention in both adult and aged rats. In addition, we tested the impact of visual distraction on performance in this task and the ability of MPH to mitigate the effects of distraction. Adult (6-12 months) and aged (18-22 months) male Sprague-Dawley rats were given oral MPH, and their cognitive and motor abilities were tested. Results suggest that while MPH improves task performance in adults; there is no improvement in the aged animals. These outcomes suggest that the use of MPH for cognitive enhancement in elderly individuals may be ineffective. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Development of hyper osmotic resistant CHO host cells for enhanced antibody production.

    Science.gov (United States)

    Kamachi, Yasuharu; Omasa, Takeshi

    2018-04-01

    Cell culture platform processes are generally employed to shorten the duration of new product development. A fed-batch process with continuous feeding is a conventional platform process for monoclonal antibody production using Chinese hamster ovary (CHO) cells. To establish a simplified platform process, the feeding method can be changed from continuous feed to bolus feed. However, this change induces a rapid increase of osmolality by the bolus addition of nutrients. The increased osmolality suppresses cell culture growth, and the final product concentration is decreased. In this study, osmotic resistant CHO host cells were developed to attain a high product concentration. To establish hyper osmotic resistant CHO host cells, CHO-S host cells were passaged long-term in a hyper osmotic basal medium. There were marked differences in cell growth of the original and established host cells under iso- (328 mOsm/kg) or hyper-osmolality (over 450 mOsm/kg) conditions. Cell growth of the original CHO host cells was markedly decreased by the induction of osmotic stress, whereas cell growth of the hyper osmotic resistant CHO host cells was not affected. The maximum viable cell concentration of hyper osmotic resistant CHO host cells was 132% of CHO-S host cells after the induction of osmotic stress. Moreover, the hyper osmotic resistant characteristic of established CHO host cells was maintained even after seven passages in iso-osmolality basal medium. The use of hyper osmotic resistance CHO host cells to create a monoclonal antibody production cell line might be a new approach to increase final antibody concentrations with a fed-batch process. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  9. Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site

    Energy Technology Data Exchange (ETDEWEB)

    Wibmer, Constantinos Kurt; Gorman, Jason; Anthony, Colin S.; Mkhize, Nonhlanhla N.; Druz, Aliaksandr; York, Talita; Schmidt, Stephen D.; Labuschagne, Phillip; Louder, Mark K.; Bailer, Robert T.; Karim, Salim S. Abdool; Mascola, John R.; Williamson, Carolyn; Moore, Penny L.; Kwong, Peter D.; Morris, Lynn (NHLS-South Africa); (NIH); (Witwatersrand); (KwaZulu-Natal)

    2016-08-31

    ABSTRACT

    All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage.

    IMPORTANCEThe conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of

  10. Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site.

    Science.gov (United States)

    Wibmer, Constantinos Kurt; Gorman, Jason; Anthony, Colin S; Mkhize, Nonhlanhla N; Druz, Aliaksandr; York, Talita; Schmidt, Stephen D; Labuschagne, Phillip; Louder, Mark K; Bailer, Robert T; Abdool Karim, Salim S; Mascola, John R; Williamson, Carolyn; Moore, Penny L; Kwong, Peter D; Morris, Lynn

    2016-11-15

    All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage. The conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to this site remain elusive

  11. Dengue virus cell entry : Unraveling the role of antibodies, maturation status, and antiviral drugs

    NARCIS (Netherlands)

    Ayala Nunez, Vanesa

    2014-01-01

    Antibody-dependent enhancement (ADE) is thought to play a critical role in the exacerbation of dengue virus-induced disease during a heterologous re-infection. Pre-existing cross-reactive anti-dengue antibodies are generally believed to bind to the newly infecting DENV and target the antibody-virus

  12. Effects of ADAM10 and ADAM17 Inhibitors on Natural Killer Cell Expansion and Antibody-dependent Cellular Cytotoxicity Against Breast Cancer CellsIn Vitro.

    Science.gov (United States)

    Pham, Dang-Huan; Kim, Ju-Sun; Kim, Sang-Ki; Shin, Dong-Jun; Uong, Nguyen-Thanh-Tung; Hyun, Hoon; Yoon, Mee Sun; Kang, Sin Jae; Ryu, Young Jae; Cho, Jin Seong; Yoon, Jung Han; Lee, Ji Shin; Cho, Duck; Lee, Soo-Hyeon; Park, Min Ho

    2017-10-01

    The inhibition of a disintegrin and metalloproteinase (ADAM) has the potential to become a novel approach for natural killer (NK) cell-based cancer immunotherapy. Thus, the aim of this study was to investigate the influence of ADAM10 and ADAM17 inhibitors on expanded NK cell to enhance antibody-dependent cellular cytotoxicity (ADCC) in breast cancer cell lines. NK cells were expanded in medium supplemented with an ADAM10 or ADAM17 inhibitor to prevent the shedding of soluble CD16/FcγRIII. The expression level of CD16 and production of interferon-gamma (IFN-γ) was detected by flow cytometry using specific antibodies. ADCC activity of expanded NK cells was estimated in trastuzumab treated breast cancer cell lines such as MCF-7, MDA-MB-231, SKBR3, and BT-474 cells. The ADAM17 inhibitor increased the purity of expanded NK cells to 90% after 14 days at 5 and 10 μM in vitro (p=0.043). However, the expansion rate of NK cells was decreased at 10 μM of the ADAM 17 inhibitor (p=0.043). Inhibition of ADAM10 suppressed the expansion of NK cells, although the NK purity was increased at 1 μM of the inhibitor. The expression of CD16 was significantly increased at 1 and 5 μM of the ADAM17 inhibitor (p=0.046, 0.028, respectively) during the culturing period. Inhibition of ADAM10 reduced the expression of CD16 on NK cells. The cytotoxic activity of the ADAM17 inhibitor treated NK cells against MCF-7 (p=0.039) and BT-474 (p=0.027) cells was significantly elevated. The ADCC activity of NK cells treated with 5 μM of ADAM17 inhibitor was significantly increased against SKBR-3 and BT-474 (p=0.027). Inhibition of ADAM17 increased the production of IFN-γ in expanded NK cells. The inhibition of ADAM17 enhanced the purity of expanded NK cells and the ADCC activity of these cells against trastuzumab treated breast cancer cell lines. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  13. A novel variable antibody fragment dimerized by leucine zippers with enhanced neutralizing potency against rabies virus G protein compared to its corresponding single-chain variable antibody fragment.

    Science.gov (United States)

    Li, Zhuang; Cheng, Yue; Xi, Hualong; Gu, Tiejun; Yuan, Ruosen; Chen, Xiaoxu; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2015-12-01

    Fatal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragments (scFv), which are composed of a variable heavy chain (VH) and a variable light chain (VL) connected by a peptide linker, can potentially be used to replace RIG. However, in our previous study, a scFv (scFV57S) specific for the rabies virus (RV) G protein showed a lower neutralizing potency than that of its parent IgG due to lower stability and altered peptide assembly pattern. In monoclonal antibodies, the VH and VL interact non-covalently, while in scFvs the VH is connected covalently with the VL by the artificial linker. In this study, we constructed and expressed two peptides 57VL-JUN-HIS and 57VH-FOS-HA in Escherichia coli. The well-known Fos and Jun leucine zippers were utilized to dimerize VH and VL similarly to the IgG counterpart. The two peptides assembled to form zipFv57S in vitro. Due to the greater similarity in structure with IgG, the zipFv57S protein showed a higher binding ability and affinity resulting in notable improvement of in vitro neutralizing activity over its corresponding scFv. The zipFv57S protein was also found to be more stable and showed similar protective rate as RIG in mice challenged with a lethal dose of RV. Our results not only indicated zipFv57S as an ideal alternative for RIG in PEP but also offered a novel and efficient hetero-dimerization pattern of VH and VL leading to enhanced neutralizing potency. Copyright © 2015. Published by Elsevier Ltd.

  14. Tyrosine kinase inhibitors as modulators of trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity in breast cancer cell lines.

    Science.gov (United States)

    Collins, Denis M; Gately, Kathy; Hughes, Clare; Edwards, Connla; Davies, Anthony; Madden, Stephen F; O'Byrne, Kenneth J; O'Donovan, Norma; Crown, John

    2017-09-01

    Trastuzumab is an anti-HER2 monoclonal antibody (mAb) therapy capable of antibody-dependent cell-mediated cytotoxicity (ADCC) and used in the treatment of HER2+ breast cancer. Through interactions with FcƴR+ immune cell subsets, trastuzumab functions as a passive immunotherapy. The EGFR/HER2-targeting tyrosine kinase inhibitor (TKI) lapatinib and the next generation TKIs afatinib and neratinib, can alter HER2 levels, potentially modulating the ADCC response to trastuzumab. Using LDH-release assays, we investigated the impact of antigen modulation, assay duration and peripheral blood mononuclear cell (PBMC) activity on trastuzumab-mediated ADCC in breast cancer models of maximal (SKBR3) and minimal (MCF-7) target antigen expression to determine if modulating the ADCC response to trastuzumab using TKIs may be a viable approach for enhancing tumor immune reactivity. HER2 levels were determined in lapatinib, afatinib and neratinib-treated SKBR3 and MCF-7 using high content analysis (HCA). Trastuzumab-mediated ADCC was assessed following treatment with TKIs utilising a colorimetric LDH release-based protocol at 4 and 12h timepoints. PBMC activity was assessed against non-MHC-restricted K562 cells. A flow cytometry-based method (CFSE/7-AAD) was also used to measure trastuzumab-mediated ADCC in medium-treated SKBR3 and MCF-7. HER2 antigen levels were significantly altered by the three TKIs in both cell line models. The TKIs significantly reduced LDH levels directly in SKBR3 cells but not MCF-7. Lapatinib and neratinib augment trastuzumab-related ADCC in SKBR3 but the effect was not consistent with antigen expression levels and was dependent on volunteer PBMC activity (vs. K562). A 12h assay timepoint produced more consistent results. Trastuzumab-mediated ADCC (PBMC:target cell ratio of 10:1) was measured at 7.6±4.7% (T12) by LDH assay and 19±3.2 % (T12) using the flow cytometry-based method in the antigen-low model MCF-7. In the presence of effector cells with high

  15. IL-15 enhances cross-reactive antibody recall responses to seasonal H3 influenza viruses in vitro [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Junqiong Huang

    2017-11-01

    Full Text Available Background: Recently, several human monoclonal antibodies that target conserved epitopes on the stalk region of influenza hemagglutinin (HA have shown broad reactivity to influenza A subtypes. Also, vaccination with recombinant chimeric HA or stem fragments from H3 influenza viruses induce broad immune protection in mice and humans. However, it is unclear whether stalk-binding antibodies can be induced in human memory B cells by seasonal H3N2 viruses. Methods: In this study, we recruited 13 donors previously exposed to H3 viruses, the majority (12 of 13 of which had been immunized with seasonal influenza vaccines. We evaluated plasma baseline strain-specific and stalk-reactive anti-HA antibodies and B cell recall responses to inactivated H3N2 A/Victoria/361/2011 virus in vitro using a high throughput multiplex (mPlex-Flu assay. Results: Stalk-reactive IgG was detected in the plasma of 7 of the subjects. Inactivated H3 viral particles rapidly induced clade cross-reactive antibodies in B cell cultures derived from all 13 donors. In addition, H3 stalk-reactive antibodies were detected in culture supernatants from 7 of the 13 donors (53.8%.  H3 stalk-reactive antibodies were also induced by H1 and H7 subtypes. Interestingly, broadly cross-reactive antibody recall responses to H3 strains were also enhanced by stimulating B cells in vitro with CpG2006 ODN in the presence of IL-15. H3 stalk-reactive antibodies were detected in  CpG2006 ODN + IL-15 stimulated B cell cultures derived from 12 of the 13 donors (92.3%, with high levels detected in cultures from 7 of the 13 donors. Conclusions: Our results demonstrate that stalk-reactive antibody recall responses induced by seasonal H3 viruses and CpG2006 ODN can be enhanced by IL-15.

  16. Speaker-dependent Dictionary-based Speech Enhancement for Text-Dependent Speaker Verification

    DEFF Research Database (Denmark)

    Thomsen, Nicolai Bæk; Thomsen, Dennis Alexander Lehmann; Tan, Zheng-Hua

    2016-01-01

    The problem of text-dependent speaker verification under noisy conditions is becoming ever more relevant, due to increased usage for authentication in real-world applications. Classical methods for noise reduction such as spectral subtraction and Wiener filtering introduce distortion and do not p...... on dictionary-based noise reduction and compare it to the baseline methods....

  17. Fab fragments of ovine antibody to colchicine enhance its clearance in the rat.

    Science.gov (United States)

    Peake, Philip W; Pianta, Timothy J; Succar, Lena; Fernando, Mangalee; Buckley, Nicholas A; Endre, Zoltan H

    2015-06-01

    Colchicine is an anti-inflammatory alkaloid used for the treatment of acute gout, but has a narrow therapeutic index. Colchicine overdoses are relatively rare, but have high mortality requiring rapid treatment. To evaluate the ability of a newly available ovine fragment antigen-binding (Fab) antibody to colchicine (ColchiFab(™)) to protect rats against renal and other injury 24 h after colchicine ingestion. Rats were gavaged with colchicine (5 mg/kg), then 2 h later injected intraperitoneally with 5 ml of sterile saline, or Fab anti-colchicine, a newly available ovine antibody to colchicine. Samples of blood were taken at 1, 2, 5 and 24 h after gavage, and urine was collected from 5 to 24 h after gavage. Concentrations of colchicine in tissue, blood and urine were measured by liquid chromatography/mass spectrometry, concentrations of Fab anti-colchicine, urinary neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 or KIM-1 by enzyme-linked immunosorbent assay or ELISA, while concentrations of creatine kinase and creatinine (Cr) were measured enzymatically. Colchicine equilibrated rapidly throughout the body and increased serum creatine kinase. Fab anti-colchicine also rapidly redistributed to the blood and remained at high concentrations over 24 h. Fab anti-colchicine caused a rapid 7.1-fold increase in serum colchicine level, followed by excretion of both colchicine and Fab anti-colchicine through the urine. This was associated with the accumulation of colchicine in the kidney, a reversal of colchicine-induced diarrhoea, and increasing urinary NGAL level; from 168 ± 48 to 477 ± 255 ng/mmol Cr [mean ± standard deviation or SD]. Fab anti-colchicine greatly increased the clearance of colchicine, although increasing NGAL level suggested the presence of mild kidney damage. These data suggest clinical utility for Fab anti-colchicine in the treatment of colchicine overdose.

  18. Activatable clinical fluorophore-quencher antibody pairs as dual molecular probes for the enhanced specificity of image-guided surgery

    Science.gov (United States)

    Obaid, Girgis; Spring, Bryan Q.; Bano, Shazia; Hasan, Tayyaba

    2017-12-01

    The emergence of fluorescently labeled therapeutic antibodies has given rise to molecular probes for image-guided surgery. However, the extraneous interstitial presence of an unbound and nonspecifically accumulated probe gives rise to false-positive detection of tumor tissue and margins. Thus, the concept of tumor-cell activation of smart probes provides a potentially superior mechanism of delineating tumor margins as well as small tumor deposits. The combination of molecular targeting with intracellular activation circumvents the presence of extracellular, nonspecific signals of targeted probe accumulation. Here, we present a demonstration of the clinical antibodies cetuximab (cet, anti-EGFR mAb) and trastuzumab (trast, anti-HER-2 mAb) conjugated to Alexa Fluor molecules and IRDye QC-1 quencher optimized at the ratio of 1∶2∶6 to provide the greatest degree of proteolytic fluorescence activation, synonymous with intracellular lysosomal degradation. The cet-AF-Q-C1 conjugate (1∶2∶6) provides up to 9.8-fold proteolytic fluorescence activation. By preparing a spectrally distinct, irrelevant sham IgG-AF-QC-1 conjugate, a dual-activatable probe approach is shown to enhance the specificity of imaging within an orthotopic AsPC-1 pancreatic cancer xenograft model. The dual-activatable approach warrants expedited clinical translation to improve the specificity of image-guided surgery by spectrally decomposing specific from nonspecific probe accumulation, binding, and internalization.

  19. Anti-Glycoprotein G Antibodies of Herpes Simplex Virus 2 Contribute to Complete Protection after Vaccination in Mice and Induce Antibody-Dependent Cellular Cytotoxicity and Complement-Mediated Cytolysis

    Directory of Open Access Journals (Sweden)

    Staffan Görander

    2014-11-01

    Full Text Available We investigated the role of antibodies against the mature portion of glycoprotein G (mgG-2 of herpes simplex virus 2 (HSV-2 in protective immunity after vaccination. Mice were immunized intramuscularly with mgG-2 and oligodeoxynucleotides containing two CpG motifs plus alum as adjuvant. All C57BL/6 mice survived and presented no genital or systemic disease. High levels of immunoglobulin G subclass 1 (IgG1 and IgG2 antibodies were detected and re-stimulated splenic CD4+ T cells proliferated and produced IFN-γ. None of the sera from immunized mice exhibited neutralization, while all sera exerted antibody-dependent cellular cytotoxicity (ADCC and complement-mediated cytolysis (ACMC activity. Passive transfer of anti-mgG-2 monoclonal antibodies, or immune serum, to naive C57BL/6 mice did not limit disease progression. Immunized B‑cell KO mice presented lower survival rate and higher vaginal viral titers, as compared with vaccinated B-cell KO mice after passive transfer of immune serum and vaccinated C57BL/6 mice. Sera from mice that were vaccinated subcutaneously and intranasally with mgG-2 presented significantly lower titers of IgG antibodies and lower ADCC and ACMC activity. We conclude that anti-mgG-2 antibodies were of importance to limit genital HSV‑2 infection. ADCC and ACMC activity are potentially important mechanisms in protective immunity, and could tentatively be evaluated in future animal vaccine studies and in clinical trials.

  20. Anti-glycoprotein g antibodies of herpes simplex virus 2 contribute to complete protection after vaccination in mice and induce antibody-dependent cellular cytotoxicity and complement-mediated cytolysis.

    Science.gov (United States)

    Görander, Staffan; Ekblad, Maria; Bergström, Tomas; Liljeqvist, Jan-Åke

    2014-11-12

    We investigated the role of antibodies against the mature portion of glycoprotein G (mgG-2) of herpes simplex virus 2 (HSV-2) in protective immunity after vaccination. Mice were immunized intramuscularly with mgG-2 and oligodeoxynucleotides containing two CpG motifs plus alum as adjuvant. All C57BL/6 mice survived and presented no genital or systemic disease. High levels of immunoglobulin G subclass 1 (IgG1) and IgG2 antibodies were detected and re-stimulated splenic CD4+ T cells proliferated and produced IFN-γ. None of the sera from immunized mice exhibited neutralization, while all sera exerted antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytolysis (ACMC) activity. Passive transfer of anti-mgG-2 monoclonal antibodies, or immune serum, to naive C57BL/6 mice did not limit disease progression. Immunized B‑cell KO mice presented lower survival rate and higher vaginal viral titers, as compared with vaccinated B-cell KO mice after passive transfer of immune serum and vaccinated C57BL/6 mice. Sera from mice that were vaccinated subcutaneously and intranasally with mgG-2 presented significantly lower titers of IgG antibodies and lower ADCC and ACMC activity. We conclude that anti-mgG-2 antibodies were of importance to limit genital HSV‑2 infection. ADCC and ACMC activity are potentially important mechanisms in protective immunity, and could tentatively be evaluated in future animal vaccine studies and in clinical trials.

  1. CD97 antibody depletes granulocytes in mice under conditions of acute inflammation via a Fc receptor-dependent mechanism

    NARCIS (Netherlands)

    Veninga, Henrike; de Groot, Dorien M.; McCloskey, Natalie; Owens, Bronwyn M.; Dessing, Mark C.; Verbeek, J. Sjef; Nourshargh, Sussan; van Eenennaam, Hans; Boots, Annemieke M.; Hamann, Jörg

    2011-01-01

    Antibodies to the pan-leukocyte adhesion-GPCR CD97 efficiently block neutrophil recruitment in mice, thereby reducing antibacterial host defense, inflammatory disease, and hematopoietic stem cell mobilization. Here, we investigated the working mechanism of the CD97 antibody 1B2. Applying sterile

  2. Breadth of HIV-1 Env-specific antibody-dependent cellular cytotoxicity: relevance to global HIV vaccine design.

    Science.gov (United States)

    Madhavi, Vijaya; Wren, Leia H; Center, Rob J; Gonelli, Christopher; Winnall, Wendy R; Parsons, Matthew S; Kramski, Marit; Kent, Stephen J; Stratov, Ivan

    2014-08-24

    The objective of this study is to determine the breadth of HIV-1 Env-specific antibody-dependent cellular cytotoxicity (ADCC) in HIV controllers and HIV progressors with a view to design globally relevant HIV vaccines. The breadth of ADCC towards four major HIV-1 Env subtypes was measured in vitro for 11 HIV controllers and 11 HIV progressors. Plasma from 11 HIV controllers (including long-term slow progressors, viremic controllers, elite controller and posttreatment controller) and 11 HIV progressors, mostly infected with HIV-1 subtype B, was analysed for ADCC responses. ADCC assays were performed against 10 HIV-1 gp120 and 8 gp140 proteins from four major HIV-1 subtypes (A, B, C and E) and 3 glycosylation-mutant gp140 proteins. ADCC-mediated natural killer cell activation was significantly broader (P = 0.02) and of higher magnitude (P HIV controllers than in HIV progressors. HIV controllers also showed significantly higher magnitude of ADCC-mediated killing of Env-coated target cells than HIV progressors to both HIV-1 subtype B and the heterologous subtype E gp140 (P = 0.001). We found good ADCC reactivity to subtype B and E Envs, less cross-reactivity to subtype A and minimal cross-reactivity to subtype C Envs. Glycosylation-dependent ADCC epitopes comprise a significant proportion of the total Env-specific ADCC response, as evident from the reduction in ADCC to nonglycosylated form of HIV-1 gp140 (P = 0.004). HIV controllers have robust ADCC responses that recognize a broad range of HIV-1 Env. Glycosylation of Env was found to be important for recognition of ADCC epitopes. Identifying conserved ADCC epitopes will assist in designing globally relevant ADCC-based HIV vaccines.

  3. Squalene-containing licensed adjuvants enhance strain-specific antibody responses against the influenza hemagglutinin and induce subtype-specific antibodies against the neuraminidase.

    Science.gov (United States)

    Schmidt, Rebecca; Holznagel, Edgar; Neumann, Britta; Alex, Nina; Sawatsky, Bevan; Enkirch, Theresa; Pfeffermann, Kristin; Kruip, Carina; von Messling, Veronika; Wagner, Ralf

    2016-10-17

    While seasonal influenza vaccines are usually non-adjuvanted, H1N1pdm09 vaccines were formulated with different squalene-containing adjuvants, to enable the reduction of antigen content thus increasing the number of doses available. To comparatively assess the effects of these adjuvants on antibody responses against matched and mismatched strains, and to correlate antibody levels with protection from disease, ferrets were immunized with 2μg of commercial H1N1pdm09 vaccine antigen alone or formulated with different licensed adjuvants. The use of squalene-containing adjuvants increased neutralizing antibody responses around 100-fold, and resulted in a significantly reduced viral load after challenge with a matched strain. While all animals mounted strong total antibody responses against the homologous H1N1 hemagglutinin (HA) protein, which correlated with the respective neutralizing antibody titers, no reactivity with the divergent H3, H5, H7, and H9 proteins were detected. Only the adjuvanted vaccines also induced antibodies against the neuraminidase (NA) protein, which were able to also recognize NA proteins from other N1 carrying strains. These findings not only support the use of squalene-containing adjuvants in dose-sparing strategies but also support speculations that the induction of NA-specific responses associated with the use of these adjuvants may confer partial protection to heterologous strains carrying the same NA subtype. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Antibody-dependent cell-mediated cytotoxicity can be induced by MUC1 peptide vaccination of breast cancer patients.

    Science.gov (United States)

    Snijdewint, F G; von Mensdorff-Pouilly, S; Karuntu-Wanamarta, A H; Verstraeten, A A; Livingston, P O; Hilgers, J; Kenemans, P

    2001-07-01

    Human polymorphic epithelial mucin (PEM, MUC1) is a high molecular weight transmembrane glycoprotein expressed on the apical cell surface of glandular epithelium and is over-expressed and hypo-glycosylated in adenocarcinomas. The extracellular part of the molecule consists mainly of a variable number of 20 amino acid repeats that contain cryptic epitopes exposed in malignancy. The objective of our study was to determine whether humanized MUC1 MAbs and Abs induced by vaccination of breast cancer patients with MUC1 peptides can effect an antibody-dependent cell-mediated cytotoxicity (ADCC). An in vitro assay has been set up in which the breast tumor cell line ZR-75-1 is used as target and PBMC of healthy donors as effector cells. Different target and effector cells, as well as various MUC1 MAbs were tested to optimize the efficacy of the in vitro assay. The humanized MAb HuHMFG-1, which recognizes the PDTR sequence in the MUC1 tandem repeat, induced a strong cell-mediated cytotoxicity. Nine MUC1-expressing tumor cell lines, including 3 bone marrow-derived cell lines, as well as 2 MUC1-transfected cell lines were susceptible to different extent to MUC1 Ab-dependent killing. Large variations in the killing capacity of PBMC from healthy donors were found. The NK cells were the essential effector cells for the MUC1 Ab-dependent killing. Plasma samples with induced high levels of MUC1 Ab were obtained from breast cancer patients repeatedly immunized with a KLH-conjugated 33-mer or 106-mer MUC1 tandem repeat. Pre- and post-vaccinated plasma samples of these patients were compared in the ADCC assay and it could be clearly demonstrated that the induced MUC1 Abs can effect tumor cell killing. MUC1 Ab-dependent cell-mediated tumor cell killing may occur in vivo and the ADCC assay can be applied to monitor MUC1 vaccination trials. Copyright 2001 Wiley-Liss, Inc.

  5. Effect of anti-carbohydrate antibodies on HIV infection in a monocytic cell line (U937)

    DEFF Research Database (Denmark)

    Hansen, J E; Nielsen, C; Clausen, H

    1991-01-01

    Monoclonal antibodies (mAbs) against carbohydrate epitopes of gp120 have recently been found to inhibit HIV infection of lymphocytes in vitro thereby opening new possibilities for vaccine considerations. Antibody-dependent enhancement of infection has however come increasingly into focus. This st......Monoclonal antibodies (mAbs) against carbohydrate epitopes of gp120 have recently been found to inhibit HIV infection of lymphocytes in vitro thereby opening new possibilities for vaccine considerations. Antibody-dependent enhancement of infection has however come increasingly into focus...... enhanced infection. This enhancement was independent of complement and could be blocked by mAb Leu3a against the CD4-receptor. The study indicated that enhancement of infection in monocytic cells can occur by the same anti-carbohydrate antibodies that neutralize infection in lymphocytes, and that antibody...

  6. β2-Glycoprotein I-Dependent Anti-Cardiolipin Antibodies Associated With Periodontitis in Patients With Systemic Lupus Erythematosus.

    Science.gov (United States)

    Wang, Chen-Ying; Chyuan, I-Tsu; Wang, Ya-Li; Kuo, Mark Yen-Ping; Chang, Ching-Wen; Wu, King-Jean; Hsu, Ping-Ning; Nagasawa, Toshiyuki; Wara-aswapati, Nawarat; Chen, Yi-Wen

    2015-08-01

    It was reported that patients with systemic lupus erythematosus (SLE) exhibited increased levels of anticardiolipin (anti-CL) antibodies, a class of antiphospholipid antibodies associated with thrombosis. β2-glycoprotein I (β2GPI) has been considered as the actual target antigen for anti-CL antibodies. This study investigates the association of periodontal infection with anti-CL antibodies in patients with SLE. Fifty-three SLE female patients and 56 healthy female volunteers were recruited in this case-control study. All participants received periodontal examinations. The presence of Porphyromonas gingivalis and Treponema denticola in saliva and plaque samples was detected by polymerase chain reaction. Levels of serum anti-CL and anti-β2GPI antibodies were examined using enzyme-linked immunosorbent assay. Patients with SLE exhibited more periodontal attachment loss and increased titers of serum anti-CL and anti-β2GPI antibodies compared with healthy controls. Patients with active SLE who harbored P. gingivalis or P. gingivalis together with T. denticola intraorally exhibited significantly higher anti-CL and anti-β2GPI antibodies than those without these bacteria. Anti-CL and anti-β2GPI antibody levels correlated positively with clinical attachment level. Furthermore, increased anti-β2GPI antibody levels were significantly associated with C-reactive protein and erythrocyte sedimentation rate. Elevated anti-CL and anti-β2GPI antibody levels were associated with periodontopathic bacteria and periodontal breakdown in patients with SLE. Periodontitis might be a modifiable risk factor for SLE.

  7. Novel antibody conjugates for enhanced tumor uptake. Final report, September 1, 1992--August 31, 1997

    International Nuclear Information System (INIS)

    Thakur, M.

    1997-01-01

    Progress in three areas of research is summarized. These are as follows: Labeling Monoclonal antibodies (MAbs) with Tc-99m and Re-186; human melanoma tumors and specific MAbs; evaluation of biological response modifiers (BRM). The techniques of labeling MAbs (IgM, IgG, F(ab') 2 or F(ab')) with Tc-99m was developed in the author's laboratory in 1989 and that with Re-186 in 1992. The techniques are in daily use in the laboratory since then and are adapted to a convenient kit formulation. The metal ions are bound at MAb sulfhydryls generated by a controlled reduction of a pair of disulfide groups. At least two types of MAbs labeled with Tc-99m by this method have been administered into patients and excellent diagnostic results have been obtained. Over the past two and a half years the author has been successfully growing human melanoma tumors in athymic Balb/c nude mice. The cell LINE, WM-9, was obtained from Dr. D Herlyn's laboratory at Wistar Institute in Philadelphia. Sufficient quantities of antihuman melanoma specific antibodies ME 31.3 (Wistar, IgG-1) and MEM-136 (Hybritech, IgG-2A) and their F(ab') 2 fragments are also available in the laboratory. The use of BRM is a rapidly evolving field. Over the past four years, the author has evaluated a number of BRMs in a quest for agents that may augment MAb tumor uptake. These included interferon-α; a pokeweed mitogen and Ukrain, an alkaloid separated from a plant Chelideonium Majis. In these preliminary studies, normal Balb/c mice were used and the BRMs were given i.p. one hour prior to the i.v. administration of tumor necrosis factor or an MAb (TNT-F(ab') 2 ) labeled with Tc-99m which served as an imaging agent. Animals were sacrificed at 1.5 hr or 4 hrs post-injection. Highlights of the work are given here in a table

  8. Hemolytic disease of the fetus and newborn due to anti-Ge3: combined antibody-dependent hemolysis and erythroid precursor cell growth inhibition.

    Science.gov (United States)

    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.

  9. Discovery of Peptidomimetic Antibody-Drug Conjugate Linkers with Enhanced Protease Specificity.

    Science.gov (United States)

    Wei, BinQing; Gunzner-Toste, Janet; Yao, Hui; Wang, Tao; Wang, Jing; Xu, Zijin; Chen, Jinhua; Wai, John; Nonomiya, Jim; Tsai, Siao Ping; Chuh, Josefa; Kozak, Katherine R; Liu, Yichin; Yu, Shang-Fan; Lau, Jeff; Li, Guangmin; Phillips, Gail D; Leipold, Doug; Kamath, Amrita; Su, Dian; Xu, Keyang; Eigenbrot, Charles; Steinbacher, Stefan; Ohri, Rachana; Raab, Helga; Staben, Leanna R; Zhao, Guiling; Flygare, John A; Pillow, Thomas H; Verma, Vishal; Masterson, Luke A; Howard, Philip W; Safina, Brian

    2018-02-08

    Antibody-drug conjugates (ADCs) have become an important therapeutic modality for oncology, with three approved by the FDA and over 60 others in clinical trials. Despite the progress, improvements in ADC therapeutic index are desired. Peptide-based ADC linkers that are cleaved by lysosomal proteases have shown sufficient stability in serum and effective payload-release in targeted cells. If the linker can be preferentially hydrolyzed by tumor-specific proteases, safety margin may improve. However, the use of peptide-based linkers limits our ability to modulate protease specificity. Here we report the structure-guided discovery of novel, nonpeptidic ADC linkers. We show that a cyclobutane-1,1-dicarboxamide-containing linker is hydrolyzed predominantly by cathepsin B while the valine-citrulline dipeptide linker is not. ADCs bearing the nonpeptidic linker are as efficacious and stable in vivo as those with the dipeptide linker. Our results strongly support the application of the peptidomimetic linker and present new opportunities for improving the selectivity of ADCs.

  10. Proteolytic detergent enzymes enhance the allergic antibody responses of guinea pigs to nonproteolytic detergent enzymes in a mixture: implications for occupational exposure.

    Science.gov (United States)

    Sarlo, K; Ritz, H L; Fletcher, E R; Schrotel, K R; Clark, E D

    1997-10-01

    A guinea pig intratracheal test was developed to assess the respiratory allergenicity of enzymes used in the detergent industry. Information gained from this test was used in a process for setting operational exposure guidelines to protect worker health. Mixtures of enzyme proteins were given to guinea pigs once per week for 10 weeks to determine whether there were interactions among enzymes that affected the induction of antibody responses to each enzyme in the mixture. Passive cutaneous anaphylaxis antibody titers against each enzyme were measured in sera. Mixtures of two or three enzymes always consisted of a protease (Alcalase, Savinase; Novo Industri A/S) with an alpha-amylase (Termamyl; Novo Industri A/S), a lipase (Lipolase; Novo Industri A/S), or both. Control animals were exposed to single enzymes. The antibody titers to Termamyl and Lipolase were significantly greater in animals dosed with the protease-containing mixtures as compared with control animals dosed with a single enzyme. Antibody titers to the protease were unchanged in the presence of additional enzymes in the mixture. Complete inactivation of protease activity abrogated the enhanced antibody response to Lipolase. Inhalation exposure of guinea pigs to a mixture of Alcalase and Lipolase also resulted in higher antibody titers to Lipolase as compared with animals exposed by inhalation to Lipolase alone, showing that the enhanced response was not due to intratracheal delivery of antigen to the respiratory tract. These results show that proteolytic enzymes in a mixture enhance antibody responses to other enzymes. This should be considered when defining exposure guidelines for protease-containing enzyme mixtures.

  11. Enhanced CDC of B cell chronic lymphocytic leukemia cells mediated by rituximab combined with a novel anti-complement factor H antibody.

    Directory of Open Access Journals (Sweden)

    Mark T Winkler

    Full Text Available Rituximab therapy for B cell chronic lymphocytic leukemia (B-CLL has met with mixed success. Among several factors to which resistance can be attributed is failure to activate complement dependent cytotoxicity (CDC due to protective complement regulatory proteins, including the soluble regulator complement factor H (CFH. We hypothesized that rituximab killing of non-responsive B-CLL cells could be augmented by a novel human monoclonal antibody against CFH. The B cells from 11 patients with B-CLL were tested ex vivo in CDC assays with combinations of CFH monoclonal antibody, rituximab, and a negative control antibody. CDC of rituximab non-responsive malignant B cells from CLL patients could in some cases be augmented by the CFH monoclonal antibody. Antibody-mediated cytotoxicity of cells was dependent upon functional complement. In one case where B-CLL cells were refractory to CDC by the combination of rituximab plus CFH monoclonal antibody, additionally neutralizing the membrane complement regulatory protein CD59 allowed CDC to occur. Inhibiting CDC regulatory proteins such as CFH holds promise for overcoming resistance to rituximab therapy in B-CLL.

  12. Characterization of an isotype-dependent monoclonal antibody against linear neutralizing epitope effective for prophylaxis of enterovirus 71 infection.

    Directory of Open Access Journals (Sweden)

    Xiao Fang Lim

    Full Text Available BACKGROUND: Enterovirus 71 (EV71 is the main causative agent of Hand, Foot and Mouth disease (HFMD and is associated with severe neurologic complications and mortalities. At present, there is no vaccine or therapeutic available for treatment. METHODOLOGY/PRINCIPAL FINDING: In this study, we generated two mAbs, denoted as mAb 51 and 53, both targeting the same linear epitope on VP1 capsid protein, spanning amino acids 215-219. In comparison, mAb 51 belonging to isotype IgM possesses neutralizing activity in vitro, whereas, mAb 53 belonging to isotype IgG1 does not have any neutralizing ability, even towards its homologous strain. When mAb 51 at 10 µg/g of body weight was administered to the 2-week-old AG129 mice one day prior to lethal challenge, 100% in vivo passive protection was observed. In contrast, the isotype control group mice, injected with an irrelevant IgM antibody before the challenge, developed limb paralysis as early as day 6 post-infection. Histological examination demonstrated that mAb 51 was able to protect against pathologic changes such as neuropil vacuolation and neuronal loss in the spinal cord, which were typical in unprotected EV-71 infected mice. BLAST analyses of that epitope revealed that it was highly conserved among all EV71 strains, but not coxsachievirus 16 (CA16. CONCLUSION: We have defined a linear epitope within the VP1 protein and demonstrated its neutralizing ability to be isotype dependent. The neutralizing property and highly conserved sequence potentiated the application of mAb 51 and 53 for protection against EV71 infection and diagnosis respectively.

  13. Detection of MHC class II expression on human basophils is dependent on antibody specificity but independent of atopic disposition

    DEFF Research Database (Denmark)

    Poulsen, Britta Cathrina; Poulsen, Lars K.; Jensen, Bettina M

    2012-01-01

    A debate has recently arisen as to whether murine basophils can function as antigen presenting cells in allergic inflammation. However, mouse and human basophils differ considerably, and the expression of MHC class II on human basophils has been investigated as a proxy for their capability...... of antigen presentation but conflicting results have emerged. In this technical note, we show that an antibody specific for all three MHC class II subtypes (HLA-DR, -DP, and -DQ), leads to a significantly higher amount of MHC class II+ basophils compared to antibodies specific for HLA-DR only. A significant...... difference was also observed between the HLA-DR specific antibodies, indicating that the choice of antibody is crucial. Furthermore, critical compensation was essential to avoid false HLA-DR+ basophils. Finally, we found that detection of MHC class II on human basophils was independent of atopic disposition....

  14. Probiotic Lactobacillus rhamnosus GG enhanced Th1 cellular immunity but did not affect antibody responses in a human gut microbiota transplanted neonatal gnotobiotic pig model.

    Directory of Open Access Journals (Sweden)

    Ke Wen

    Full Text Available This study aims to establish a human gut microbiota (HGM transplanted gnotobiotic (Gn pig model of human rotavirus (HRV infection and diarrhea, and to verify the dose-effects of probiotics on HRV vaccine-induced immune responses. Our previous studies using the Gn pig model found that probiotics dose-dependently regulated both T cell and B cell immune responses induced by rotavirus vaccines. We generated the HGM transplanted neonatal Gn pigs through daily feeding of neonatal human fecal suspension to germ-free pigs for 3 days starting at 12 hours after birth. We found that attenuated HRV (AttHRV vaccination conferred similar overall protection against rotavirus diarrhea and virus shedding in Gn pigs and HGM transplanted Gn pigs. HGM promoted the development of the neonatal immune system, as evidenced by the significantly enhanced IFN-γ producing T cell responses and reduction of regulatory T cells and their cytokine production in the AttHRV-vaccinated pigs. The higher dose Lactobacillus rhamnosus GG (LGG feeding (14 doses, up to 109 colony-forming-unit [CFU]/dose effectively increased the LGG counts in the HGM Gn pig intestinal contents and significantly enhanced HRV-specific IFN-γ producing T cell responses to the AttHRV vaccine. Lower dose LGG (9 doses, up to 106 CFU/dose was ineffective. Neither doses of LGG significantly improved the protection rate, HRV-specific IgA and IgG antibody titers in serum, or IgA antibody titers in intestinal contents compared to the AttHRV vaccine alone, suggesting that an even higher dose of LGG is needed to overcome the influence of the microbiota to achieve the immunostimulatory effect in the HGM pigs. This study demonstrated that HGM Gn pig is an applicable animal model for studying immune responses to rotavirus vaccines and can be used for studying interventions (i.e., probiotics and prebiotics that may enhance the immunogenicity and protective efficacy of vaccines through improving the gut microbiota.

  15. Refining the LPS-Antigen in Salmonella Antibody Elisa for Poultry Enhanced Specificity without Impairing Sensitivity

    DEFF Research Database (Denmark)

    Lauritsen, Klara Tølbøl; Lind, Peter; Klausen, Joan

    2014-01-01

    In the Danish serological surveillance for Salmonella in poultry (serum and egg yolk) a mix-ELISA is used, based on S. typhimurium and S. enteritidis antigens (Feld et al., 2000). When we evaluated results of the test retrospectively, over the years an unacceptably large fraction of seropositive...... findings could not be confirmed by the subsequent confirmatory bacteriological sampling in the herd. Therefore we tried to enhance specificity of the ELISA, without losing sensitivity, by refining the antigens used....

  16. The chromatin Remodeler CHD8 is required for activation of progesterone receptor-dependent enhancers.

    Directory of Open Access Journals (Sweden)

    María Ceballos-Chávez

    2015-04-01

    Full Text Available While the importance of gene enhancers in transcriptional regulation is well established, the mechanisms and the protein factors that determine enhancers activity have only recently begun to be unravelled. Recent studies have shown that progesterone receptor (PR binds regions that display typical features of gene enhancers. Here, we show by ChIP-seq experiments that the chromatin remodeler CHD8 mostly binds promoters under proliferation conditions. However, upon progestin stimulation, CHD8 re-localizes to PR enhancers also enriched in p300 and H3K4me1. Consistently, CHD8 depletion severely impairs progestin-dependent gene regulation. CHD8 binding is PR-dependent but independent of the pioneering factor FOXA1. The SWI/SNF chromatin-remodelling complex is required for PR-dependent gene activation. Interestingly, we show that CHD8 interacts with the SWI/SNF complex and that depletion of BRG1 and BRM, the ATPases of SWI/SNF complex, impairs CHD8 recruitment. We also show that CHD8 is not required for H3K27 acetylation, but contributes to increase accessibility of the enhancer to DNaseI. Furthermore, CHD8 was required for RNAPII recruiting to the enhancers and for transcription of enhancer-derived RNAs (eRNAs. Taken together our data demonstrate that CHD8 is involved in late stages of PR enhancers activation.

  17. Immunohistochemical analysis of the D-type cyclin-dependent kinases Cdk4 and Cdk6, using a series of monoclonal antibodies.

    Science.gov (United States)

    Lukas, C; Jensen, S K; Bartkova, J; Lukas, J; Bartek, J

    1999-06-01

    Cellular signal transduction cascades triggered by mitogenic or antiproliferative cues eventually converge on a biochemical mechanism centered around the retinoblastoma tumor suppressor (pRb), the so-called RB pathway that governs G1-phase progression and guards the commitment to enter S phase. pRb, together with its immediate upstream regulators, the D-type cyclins, their partner cyclin-dependent kinases Cdk4 and Cdk6, and the Cdk inhibitors, form a functional unit that is involved in major decisions about cellular fate, and whose components, including the proto-oncogenic cyclin D-dependent kinases, are commonly deregulated in many types of cancer. We report here the production and characterization of a series of 12 monoclonal antibodies (MAbs) that specifically recognize either Cdk4 or Cdk6. These antibodies are proving to be invaluable molecular probes for defining abundance, subcellular localization, binding partners, and ultimately the function(s) of these cell cycle-regulatory kinases. Localization of the target epitopes was mapped by peptide enzyme-linked immunoadsorbent assay (ELISA), and two antibodies recognizing sequences adjacent to N-terminus of Cdk4 can discriminate between the wild-type protein and the oncogenic, melanoma-associated R24C mutant of this kinase. Individual antibodies of our panel recognize distinct pools of Cdk4/6, a feature reflected by their differential applicability in immunoblotting, immunoprecipitation, kinase assays, and immunostaining including immunohistochemistry on archival paraffin-embedded tissue sections. Collectively, the antibodies described in this study provide the means for immunochemical analyses of the cyclin D-dependent kinases in human and animal cells, and represent useful molecular tools that should help better understand the biological roles of Cdk4 and Cdk6 in normal cell-cycle control, and their oncogenic activity in tumor cells.

  18. Enhancement of retroviral infection in vitro by anti-Le(y) IgG: reversal by humanization of monoclonal mouse antibody

    DEFF Research Database (Denmark)

    Hansen, J E; Sørensen, A M; Arendrup, M

    1993-01-01

    with no indication of any alternative pathway of infection, as evidenced by abrogation of enhancement by anti-CD4 MAb or soluble recombinant CD4, and also the inability of anti-Le(y) MAb to mediate HIV infection of HSB-2 cells in which HTLV-1/HIV pseudovirus infection was enhanced. While F(ab)2 fragments of ABL 364...... also enhanced infection, a human/mouse chimeric antibody and a fully humanized antibody had no enhancing effect on free virus infection. We suggest that binding of anti-Le(y) ABL 364 or its F(ab)2 fragment induced a conformational change in the gp120 oligomers facilitating the process of infection...

  19. Liposome-based polymer complex as a novel adjuvant: enhancement of specific antibody production and isotype switch

    Directory of Open Access Journals (Sweden)

    Chen CH

    2012-02-01

    Full Text Available Chia-Hung Chen1,*, Yu-Ling Lin1,*, Yen-Ku Liu1, Pei-Juin He2, Ching-Min Lin1, Yi-Han Chiu2, Chang-Jer Wu3, Tian-Lu Cheng4, Shih-Jen Liu5,6,**, Kuang-Wen Liao1,2,**1Institute of Molecular Medicine and Bioengineering, 2Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, 3Department of Food Science, National Taiwan Ocean University, Keelung, 4Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, 5National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Miaoli, 6Graduate Institute of Immunology, China Medical University, Taichung, Taiwan, , *Chia-Hung Chen and Yu-Ling Lin contributed equally to this work**Kuang-Wen Liao and Shih-Jen Liu contributed equally to this workAbstract: The aim of vaccination is to induce appropriate immunity against pathogens. Antibody-mediated immunity is critical for protection against many virus diseases, although it is becoming more evident that coordinated, multifunctional immune responses lead to the most effective defense. Specific antibody (Ab isotypes are more efficient at protecting against pathogen invasion in different locations in the body. For example, compared to other Ab isotypes, immunoglobulin (Ig A provides more protection at mucosal areas. In this study, we developed a cationic lipopolymer (liposome-polyethylene glycol-polyethyleneimine complex [LPPC] adjuvant that strongly adsorbs antigens or immunomodulators onto its surface to enhance or switch immune responses. The results demonstrate that LPPC enhances uptake ability, surface marker expression, proinflammatory cytokine release, and antigen presentation in mouse phagocytes. In contrast to Freund's adjuvant, LPPC preferentially activates Th1-immunity against antigens in vivo. With lipopolysaccharides or CpG oligodeoxynucleotides, LPPC dramatically enhances the IgA or IgG2A proportion of total Ig, even in hosts that have developed

  20. Exogenous insulin-like growth factor 2 administration enhances memory consolidation and persistence in a time-dependent manner.

    Science.gov (United States)

    Lee, Younghwan; Lee, Young Woo; Gao, Qingtao; Lee, Younghwa; Lee, Hyung Eun; Ryu, Jong Hoon

    2015-10-05

    Memory consolidation is an important process for the formation of long-term memory. We have previously reported that mature brain-derived neurotrophic factor enhances memory consolidation within 9h after initial learning. Recent studies suggest that insulin-like growth factor 2 (IGF2) significantly enhances memory consolidation and prevents forgetting. Thus, we hypothesized that IGF2 exerts its activity on cognitive performance in a time-dependent manner as observed in our previous study. In the one-trial step-through inhibitory avoidance task, we demonstrate that a bilateral injection of IGF2 into the dorsal hippocampus 6 or 9 h after training significantly enhanced the step-through latencies compared with the vehicle-treated controls in the retention trial, which was conducted 24 h after the acquisition trial. However, 12h post-training, IGF2 injection did not increase the step-through latencies. Intriguingly, in the retention trial at 21 days after the training, hippocampal IGF2 injection 6, 9 or 12 h after the acquisition trial significantly increased the step-through latencies compared with the vehicle-treated controls. IGF2 administration at 9 h and 12 h after the acquisition trial significantly increased discrimination index and exploration time on the novel-located object in the test trial at 24 h and 21 days, respectively, after the acquisition trial in the novel location recognition task. In addition, IGF2-induced an increase in the step-through latencies in the retention trial 24 h or 21 days, respectively, after the initial learning was completely abolished by co-injected anti-IGF2 receptor antibody. These results suggest that IGF2 enhances memory consolidation within 9h after initial learning, and increased IGF2 within the 12 h after the acquisition trial, which represents a delayed consolidation phase, is also critical for memory persistence. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Enhancing blockade of Plasmodium falciparum erythrocyte invasion: assessing combinations of antibodies against PfRH5 and other merozoite antigens.

    Directory of Open Access Journals (Sweden)

    Andrew R Williams

    Full Text Available No vaccine has yet proven effective against the blood-stages of Plasmodium falciparum, which cause the symptoms and severe manifestations of malaria. We recently found that PfRH5, a P. falciparum-specific protein expressed in merozoites, is efficiently targeted by broadly-neutralizing, vaccine-induced antibodies. Here we show that antibodies against PfRH5 efficiently inhibit the in vitro growth of short-term-adapted parasite isolates from Cambodia, and that the EC(50 values of antigen-specific antibodies against PfRH5 are lower than those against PfAMA1. Since antibody responses elicited by multiple antigens are speculated to improve the efficacy of blood-stage vaccines, we conducted detailed assessments of parasite growth inhibition by antibodies against PfRH5 in combination with antibodies against seven other merozoite antigens. We found that antibodies against PfRH5 act synergistically with antibodies against certain other merozoite antigens, most notably with antibodies against other erythrocyte-binding antigens such as PfRH4, to inhibit the growth of a homologous P. falciparum clone. A combination of antibodies against PfRH4 and basigin, the erythrocyte receptor for PfRH5, also potently inhibited parasite growth. This methodology provides the first quantitative evidence that polyclonal vaccine-induced antibodies can act synergistically against P. falciparum antigens and should help to guide the rational development of future multi-antigen vaccines.

  2. IFN-α augments natural killer-mediated antibody-dependent cellular cytotoxicity of HIV-1-infected autologous CD4+ T cells regardless of major histocompatibility complex class 1 downregulation.

    Science.gov (United States)

    Tomescu, Costin; Tebas, Pablo; Montaner, Luis J

    2017-03-13

    We have previously shown that IFN-α stimulation augments direct natural killer (NK) cell lysis of autologous CD4 primary T cells infected with certain HIV-1 isolates based upon major histocompatibility complex class 1 (MHC-1) downregulation capacity. Here, we investigated if antibody-dependent cellular cytotoxicity (ADCC) could trigger lysis of HIV-1 isolates that were resistant to direct NK lysis and if IFN-α prestimulation of NK cells could further enhance ADCC. Using broadly neutralizing monoclonal antibodies against gp120 (VRC01 or PGV04) or plasma from HIV-1-infected patients (ART-suppressed or elite controller) to trigger ADCC, we measured NK cell chromium release cytotoxicity against HIV-1-infected autologous CD4 primary T cells and NK cell CD107a degranulation against gp120-coated CD4 T cells. Total or NK-depleted peripheral blood mononuclear cells were used as effectors in the presence or absence of IFN-α prestimulation. Plasma from HIV-1-infected patients and monoclonal antibodies against gp120 could trigger NK-dependent ADCC lysis of viral isolates that were resistant to direct NK cell lysis following IFN-α stimulation. In contrast, viral isolates that exhibited potent MHC-I downregulation capacity could be lysed by NK cells through either IFN-α stimulated direct cytotoxicity or through ADCC. When utilized in combination, IFN-α prestimulation significantly augmented ADCC lysis of HIV-1-infected target cells and increased NK cell CD107a degranulation against gp120-coated ADCC targets (P cytotoxicity depending on MHC downregulation status.

  3. eRNAs are required for p53-dependent enhancer activity and gene transcription.

    Science.gov (United States)

    Melo, Carlos A; Drost, Jarno; Wijchers, Patrick J; van de Werken, Harmen; de Wit, Elzo; Oude Vrielink, Joachim A F; Elkon, Ran; Melo, Sónia A; Léveillé, Nicolas; Kalluri, Raghu; de Laat, Wouter; Agami, Reuven

    2013-02-07

    Binding within or nearby target genes involved in cell proliferation and survival enables the p53 tumor suppressor gene to regulate their transcription and cell-cycle progression. Using genome-wide chromatin-binding profiles, we describe binding of p53 also to regions located distantly from any known p53 target gene. Interestingly, many of these regions possess conserved p53-binding sites and all known hallmarks of enhancer regions. We demonstrate that these p53-bound enhancer regions (p53BERs) indeed contain enhancer activity and interact intrachromosomally with multiple neighboring genes to convey long-distance p53-dependent transcription regulation. Furthermore, p53BERs produce, in a p53-dependent manner, enhancer RNAs (eRNAs) that are required for efficient transcriptional enhancement of interacting target genes and induction of a p53-dependent cell-cycle arrest. Thus, our results ascribe transcription enhancement activity to p53 with the capacity to regulate multiple genes from a single genomic binding site. Moreover, eRNA production from p53BERs is required for efficient p53 transcription enhancement. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Antibodies to glutamic acid decarboxylase are associated with HLA-DR genotypes in both Australians and Asians with type 1 (insulin-dependent) diabetes mellitus.

    Science.gov (United States)

    Serjeantson, S W; Kohonen-Corish, M R; Rowley, M J; Mackay, I R; Knowles, W; Zimmet, P

    1992-10-01

    Antibodies to glutamic acid decarboxylase, previously known as the 64 kD antigen, appear to be more predictive of Type 1 (insulin-dependent) diabetes mellitus in Caucasoids than other autoantibodies to islet cell antigens. However, seropositivity to glutamic acid decarboxylase is not universal at the onset of Type 1 diabetes and the prevalence in Asians is low compared to Caucasoid patients. This suggests the involvement of multiple pancreatic autoantigens in the Type 1 diabetes autoimmune process or, genetic differences within and between ethnic groups that contribute to the heterogeneous autoimmune response to glutamic acid decarboxylase or both. Alternatively some cases of Type 1 diabetes could have an aetiology unrelated to autoimmunity. This study examined the differential response to glutamic acid decarboxylase according to HLA-DR and -DQ genotypes, as determined by RFLP, in 49 white Australian and 44 Asian patients with Type 1 diabetes. Among Australians heterozygous for HLA-DR3, DR4, 85% were positive for antibodies to glutamic acid decarboxylase, significantly different (p = 0.039) from the prevalence of 48% in patients with at least one HLA-DR antigen other than DR3 or DR4. Also, among Australians, the presence of "low risk" HLA-DQ antigens, namely DQw5, DQw6 or DQw7, reduced the prevalence of antibodies to glutamic acid decarboxylase by 40% (p = 0.064). Among Asians with Type 1 diabetes and with antibodies to glutamic acid decarboxylase, HLA-DR9 was significantly (p = 0.037) increased in frequency, at 63% compared with 22% in those without glutamic acid decarboxylase antibodies, and the presence of a "low risk" HLA-DQ allele reduced the antibody rates by 87% (p = 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook; Shin, Seung-Min; Bae, Jeomil [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Dong-Myung [Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Yoo, Tae Hyeon [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Yong-Sung, E-mail: kimys@ajou.ac.kr [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of)

    2015-11-27

    Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3–4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity.

  6. Evidence that active protection following oral immunization of mice with live rotavirus is not dependent on neutralizing antibody.

    Science.gov (United States)

    Ward, R L; McNeal, M M; Sheridan, J F

    1992-05-01

    Studies were performed to determine whether active immunity against murine rotavirus (EDIM) infection of mice correlated with titers of neutralizing antibody to the challenge virus. Neonatal mice administered either murine or heterologous rotaviruses all developed diarrhea and high titers of serum rotavirus IgG. However, only mice given EDIM, the murine EB, or simian SA11-FEM strains were protected against EDIM infection when challenged 60 days later. Other serotype 3 strains (RRV, SA11-SEM), as well as strains belonging to serotypes 5 and 6 (OSU, NCDV, WC3), were not protective. Serum neutralizing antibody titers to EDIM were almost undetectable after rotavirus infection with any strain and could not, therefore, be correlated with protection. Likewise, intestinal neutralizing antibody titers were extremely low 21 days after EDIM infection, and by 60 days after inoculation, EDIM-infected mice had no greater intestinal neutralizing antibody titers than uninoculated controls. Mice inoculated with SA11-FEM as neonates had much higher serum rotavirus IgG responses than mice inoculated as adults, and only those infected with this virus as neonates were protected. Thus, although immunity to EDIM did not correlate with the presence of neutralizing antibody to EDIM, it did correlate with the overall magnitude of the immune response after inoculation with SA11-FEM.

  7. Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation.

    Science.gov (United States)

    Li, Wenbo; Notani, Dimple; Ma, Qi; Tanasa, Bogdan; Nunez, Esperanza; Chen, Aaron Yun; Merkurjev, Daria; Zhang, Jie; Ohgi, Kenneth; Song, Xiaoyuan; Oh, Soohwan; Kim, Hong-Sook; Glass, Christopher K; Rosenfeld, Michael G

    2013-06-27

    The functional importance of gene enhancers in regulated gene expression is well established. In addition to widespread transcription of long non-coding RNAs (lncRNAs) in mammalian cells, bidirectional ncRNAs are transcribed on enhancers, and are thus referred to as enhancer RNAs (eRNAs). However, it has remained unclear whether these eRNAs are functional or merely a reflection of enhancer activation. Here we report that in human breast cancer cells 17β-oestradiol (E2)-bound oestrogen receptor α (ER-α) causes a global increase in eRNA transcription on enhancers adjacent to E2-upregulated coding genes. These induced eRNAs, as functional transcripts, seem to exert important roles for the observed ligand-dependent induction of target coding genes, increasing the strength of specific enhancer-promoter looping initiated by ER-α binding. Cohesin, present on many ER-α-regulated enhancers even before ligand treatment, apparently contributes to E2-dependent gene activation, at least in part by stabilizing E2/ER-α/eRNA-induced enhancer-promoter looping. Our data indicate that eRNAs are likely to have important functions in many regulated programs of gene transcription.

  8. Maternal plasma viral load and neutralizing/enhancing antibodies in vertical transmission of HIV: A non-randomized prospective study

    Directory of Open Access Journals (Sweden)

    Weiss Heidi

    2005-02-01

    Full Text Available Abstract Background We examined the association and interaction between maternal viral load and antibodies in vertical transmission of HIV in a non-randomized prospective study of 43 HIV-1 infected pregnant women who attended the San Juan City Hospital, Puerto Rico, and their 45 newborn infants. The women and infants received antiretroviral therapy. Methods A nested PCR assay of the HIV-1 envelope V3 region and infant PBMC culture were performed to determine HIV status of the infants. Maternal and infant plasma were tested for HIV neutralization or enhancement in monocyte-derived macrophages. Results Twelve (26.7% infants were positive by the HIV V3 PCR assay and 3 of the 12 were also positive by culture. There was a trend of agreement between high maternal viral load and HIV transmission by multivariate analysis (OR = 2.5, CI = 0.92, p = 0.0681. Both maternal and infant plasma significantly (p = 0.001 for both reduced HIV replication at 10-1 dilution compared with HIV negative plasma. Infant plasma neutralized HIV (p = 0.001 at 10-2 dilution but maternal plasma lost neutralizing effect at this dilution. At 10-3 dilution both maternal and infant plasma increased virus replication above that obtained with HIV negative plasma but only the increase by maternal plasma was statistically significant (p = 0.005. There were good agreements in enhancing activity in plasma between mother-infant pairs, but there was no significant association between HIV enhancement by maternal plasma and vertical transmission. Conclusion Although not statistically significant, the trend of association between maternal viral load and maternal-infant transmission of HIV supports the finding that viral load is a predictor of maternal-infant transmission. Both maternal and infant plasma neutralized HIV at low dilution and enhanced virus replication at high dilution. The antiretroviral treatments that the women received and the small sample size may have contributed to the

  9. Discovery and Characterization of Super-Enhancer Associated Dependencies in Diffuse Large B-Cell Lymphoma

    Science.gov (United States)

    Chapuy, Bjoern; McKeown, Michael R.; Lin, Charles Y.; Monti, Stefano; Roemer, Margaretha G.M.; Qi, Jun; Rahl, Peter B.; Sun, Heather H.; Yeda, Kelly T.; Doench, John G; Reichert, Elaine; Kung, Andrew L.; Rodig, Scott J.; Young, Richard A.; Shipp, Margaret A.; Bradner, James E.

    2014-01-01

    Summary Diffuse Large B-Cell Lymphoma (DLBCL) is a biologically heterogeneous and clinically aggressive disease. Here, we explore the role of BET bromodomain proteins in DLBCL, using integrative chemical genetics and functional epigenomics. We observe highly asymmetric loading of BRD4 at enhancers, with approximately 33% of all BRD4 localizing to enhancers at 1.6% of occupied genes. These super-enhancers prove particularly sensitive to bromodomain inhibition, explaining the selective effect of BET inhibitors on oncogenic and lineage-specific transcriptional circuits. Functional study of genes marked by super-enhancers identifies DLBCLs dependent on OCA-B and suggests a strategy for discovering unrecognized cancer dependencies. Translational studies performed on a comprehensive panel of DLBCLs establish a therapeutic rationale for evaluating BET inhibitors in this disease. PMID:24332044

  10. Polarization Dependence of Surface Enhanced Raman Scattering on a Single Dielectric Nanowire

    Directory of Open Access Journals (Sweden)

    Hua Qi

    2012-01-01

    Full Text Available Our measurements of surface enhanced Raman scattering (SERS on Ga2O3 dielectric nanowires (NWs core/silver composites indicate that the SERS enhancement is highly dependent on the polarization direction of the incident laser light. The polarization dependence of the SERS signal with respect to the direction of a single NW was studied by changing the incident light angle. Further investigations demonstrate that the SERS intensity is not only dependent on the direction and wavelength of the incident light, but also on the species of the SERS active molecule. The largest signals were observed on an NW when the incident 514.5 nm light was polarized perpendicular to the length of the NW, while the opposite phenomenon was observed at the wavelength of 785 nm. Our theoretical simulations of the polarization dependence at 514.5 nm and 785 nm are in good agreement with the experimental results.

  11. Glucocorticoid enhancement of dorsolateral striatum-dependent habit memory requires concurrent noradrenergic activity.

    Science.gov (United States)

    Goodman, J; Leong, K-C; Packard, M G

    2015-12-17

    Previous findings indicate that post-training administration of glucocorticoid stress hormones can interact with the noradrenergic system to enhance consolidation of hippocampus- or amygdala-dependent cognitive/emotional memory. The present experiments were designed to extend these findings by examining the potential interaction of glucocorticoid and noradrenergic mechanisms in enhancement of dorsolateral striatum (DLS)-dependent habit memory. In experiment 1, different groups of adult male Long-Evans rats received training in two DLS-dependent memory tasks. In a cued water maze task, rats were released from various start points and were reinforced to approach a visibly cued escape platform. In a response-learning version of the water plus-maze task, animals were released from opposite starting positions and were reinforced to make a consistent egocentric body-turn to reach a hidden escape platform. Immediately post-training, rats received peripheral injections of the glucocorticoid corticosterone (1 or 3 mg/kg) or vehicle solution. In both tasks, corticosterone (3 mg/kg) enhanced DLS-dependent habit memory. In experiment 2, a separate group of animals received training in the response learning version of the water plus-maze task and were given peripheral post-training injections of corticosterone (3 mg/kg), the β-adrenoreceptor antagonist propranolol (3 mg/kg), corticosterone and propranolol concurrently, or control vehicle solution. Corticosterone injections again enhanced DLS-dependent memory, and this effect was blocked by concurrent administration of propranolol. Propranolol administration by itself (3 mg/kg) did not influence DLS-dependent memory. Taken together, the findings indicate an interaction between glucocorticoid and noradrenergic mechanisms in DLS-dependent habit memory. Propranolol administration may be useful in treating stress-related human psychopathologies associated with a dysfunctional DLS-dependent habit memory system. Copyright © 2015

  12. Time dependent effects of two absorption enhancers on the nasal absorption of growth hormone in rabbits

    DEFF Research Database (Denmark)

    Vermehren, Charlotte; Hansen, H.S.; Thomsen, M.K.

    1996-01-01

    Enhancer-based drug preparations allow absorption of peptid drugs. We investigated the reversibility with time of nasal absorption of human growth hormone (hGH) induced by the absorption enhancers didecanoylhposphatidylcholine (DDPC) and Ó-cyclodextrin (Ó-CD). Rabbits were dosed intranasally...... with enhancer in the absence of hGH at time -3, -1, -0.5 and 0 h. At time zero the same groups of rabbits were dosed with a hGH powder devoid of the enhancers. Values for plasma hGH AUC and Cmax were estimated in order to measure the degree of absorption, and rabbits receiving hGH together with enhancers were...... used as positive references. With an enhancer preparation of 8% DDPC and 30% Ó-CD, the hGH AUC and Cmax values showed a significant time-dependent decrease after enhancer administration. This may indicate recovery of the enhancer-induced mucosal leakiness as well as clearance of the enhancers from...

  13. Time dependent effects of two absorption enhancers on the nasal absorption of growth hormone in rabbits

    DEFF Research Database (Denmark)

    Vermehren, C.; Hansen, Harald S.; Thomsen, M.K.

    1996-01-01

    Enhancer-based drug preparations allow absorption of peptide drugs. We investigated the reversibility with time of nasal absorption of human growth hormone (hGH) induced by the absorption enhancers didecanoylphosphatidylcholine (DDPC) and a-cyclodextrin (a-CD). Rabbits were dosed intranasally...... with enhancer in the absence of hGH at time -3, -1, -0.5 and 0 h. At time zero the same groups of rabbits were dosed with a hGH powder devoid of the enhancers. Values for plasma hGH AUC and C(max) were estimated in order to measure the degree of absorption, and rabbits receiving hGH together with enhancers were...... used as positive references. With an enhancer preparation of 8% DDPC and 30% a-CD, the hGH AUC and C(max) values showed a significant time-dependent decrease after enhancer administration. This may indicate recovery of the enhancer-induced mucosal leakiness as well as clearance of the enhancers from...

  14. Boosting antibody responses by targeting antigens to dendritic cells.

    Science.gov (United States)

    Caminschi, Irina; Shortman, Ken

    2012-02-01

    Delivering antigens directly to dendritic cells (DCs) in situ, by injecting antigens coupled to antibodies specific for DC surface molecules, is a promising strategy for enhancing vaccine efficacy. Enhanced cytotoxic T cell responses are obtained if an adjuvant is co-administered to activate the DC. Such DC targeting is also effective at enhancing humoral immunity, via the generation of T follicular helper cells. Depending on the DC surface molecule targeted, antibody production can be enhanced even in the absence of adjuvants. In the case of Clec9A as the DC surface target, enhanced antibody production is a consequence of the DC-restricted expression of the target molecule. Few other cells absorb the antigen-antibody construct, therefore, it persists in the bloodstream, allowing sustained antigen presentation, even by non-activated DCs. Copyright © 2011 Elsevier Ltd. All rights reserved.

  15. Carrier priming or suppression: understanding carrier priming enhancement of anti-polysaccharide antibody response to conjugate vaccines.

    Science.gov (United States)

    Pobre, Karl; Tashani, Mohamed; Ridda, Iman; Rashid, Harunor; Wong, Melanie; Booy, Robert

    2014-03-14

    With the availability of newer conjugate vaccines, immunization schedules have become increasingly complex due to the potential for unpredictable immunologic interference such as 'carrier priming' and 'carrier induced epitopic suppression'. Carrier priming refers to an augmented antibody response to a carbohydrate portion of a glycoconjugate vaccine in an individual previously primed with the carrier protein. This review aims to provide a critical evaluation of the available data on carrier priming (and suppression) and conceptualize ways by which this phenomenon can be utilized to strengthen vaccination schedules. We conducted this literature review by searching well-known databases to date to identify relevant studies, then extracted and synthesized the data on carrier priming of widely used conjugate polysaccharide vaccines, such as, pneumococcal conjugate vaccine (PCV), meningococcal conjugate vaccine (MenCV) and Haemophilus influenzae type b conjugate vaccines (HibV). We found evidence of carrier priming with some conjugate vaccines, particularly HibV and PCV, in both animal and human models but controversy surrounds MenCV. This has implications for the immunogenicity of conjugate polysaccharide vaccines following the administration of tetanus-toxoid or diphtheria-toxoid containing vaccine (such as DTP). Available evidence supports a promising role for carrier priming in terms of maximizing the immunogenicity of conjugate vaccines and enhancing immunization schedule by making it more efficient and cost effective. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Combination of two anti-CD5 monoclonal antibodies synergistically induces complement-dependent cytotoxicity of chronic lymphocytic leukaemia cells

    DEFF Research Database (Denmark)

    Klitgaard, Josephine L; Koefoed, Klaus; Geisler, Christian

    2013-01-01

    The treatment of chronic lymphocytic leukaemia (CLL) has been improved by introduction of monoclonal antibodies (mAbs) that exert their effect through secondary effector mechanisms. CLL cells are characterized by expression of CD5 and CD23 along with CD19 and CD20, hence anti-CD5 Abs that engage...

  17. A dual vaccine against influenza & Alzheimer's disease failed to enhance anti-β-amyloid antibody responses in mice with pre-existing virus specific memory.

    Science.gov (United States)

    Davtyan, Hayk; Ghochikyan, Anahit; Hovakimyan, Armine; Davtyan, Arpine; Cadagan, Richard; Marleau, Annette M; Albrecht, Randy A; García-Sastre, Adolfo; Agadjanyan, Michael G

    2014-12-15

    Novel dual vaccine, WSN-Aβ(1-10), based on the recombinant influenza virus, expressing immunodominant B-cell epitope of β-amyloid, simultaneously induced therapeutically potent anti-Aβ and anti-influenza antibodies. In this study we showed that boosting of WSN-WT primed mice with WSN-Aβ(1-10) enhances anti-viral, but fails to induce anti-Aβ antibody responses. This inhibition is associated with expression of Aβ(1-10) within the context of an inactivated influenza virus vaccine. These results demonstrate that the use of an inactivated influenza virus as a carrier for AD vaccine may not be applicable due to the possible inhibition of anti-Aβ antibody response in individuals previously vaccinated or infected with influenza. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Daratumumab-mediated lysis of primary multiple myeloma cells is enhanced in combination with the human anti-KIR antibody IPH2102 and lenalidomide

    DEFF Research Database (Denmark)

    Nijhof, I. S.; Lammerts van Bueren, J. J.; van Kessel, B.

    2015-01-01

    killer cell inhibitory receptors with the human monoclonal anti-KIR antibody IPH2102, next to activation of natural killer cells with the immune modulatory drug lenalidomide. In 4-hour antibody-dependent cell-mediated cytotoxicity assays, IPH2102 did not induce lysis of multiple myeloma cell lines......RIIa-131R allele, who bind IgG1 with lower affinity than patients carrying the FcgammaRIIIa-158V allele or the FcgammaRIIa-131H allele. Finally, a further synergistically improved myeloma cell lysis with the daratumumab-IPH2102 combination was observed by adding lenalidomide, which suggests that more...

  19. Identification of antibody glycosylation structures that predict monoclonal antibody Fc-effector function.

    Science.gov (United States)

    Chung, Amy W; Crispin, Max; Pritchard, Laura; Robinson, Hannah; Gorny, Miroslaw K; Yu, Xiaojie; Bailey-Kellogg, Chris; Ackerman, Margaret E; Scanlan, Chris; Zolla-Pazner, Susan; Alter, Galit

    2014-11-13

    To determine monoclonal antibody (mAb) features that predict fragment crystalizable (Fc)-mediated effector functions against HIV. Monoclonal antibodies, derived from Chinese hamster ovary cells or Epstein-Barr virus-immortalized mouse heteromyelomas, with specificity to key regions of the HIV envelope including gp120-V2, gp120-V3 loop, gp120-CD4(+) binding site, and gp41-specific antibodies, were functionally profiled to determine the relative contribution of the variable and constant domain features of the antibodies in driving robust Fc-effector functions. Each mAb was assayed for antibody-binding affinity to gp140(SR162), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and for the ability to bind to FcγRIIa, FcγRIIb and FcγRIIIa receptors. Antibody glycan profiles were determined by HPLC. Neither the specificity nor the affinity of the mAbs determined the potency of Fc-effector function. FcγRIIIa binding strongly predicted ADCC and decreased galactose content inversely correlated with ADCP, whereas N-glycolylneuraminic acid-containing structures exhibited enhanced ADCP. Additionally, the bi-antenary glycan arm onto which galactose was added predicted enhanced binding to FcγRIIIa and ADCC activity, independent of the specificity of the mAb. Our studies point to the specific Fc-glycan structures that can selectively promote Fc-effector functions independently of the antibody specificity. Furthermore, we demonstrated antibody glycan structures associated with enhanced ADCP activity, an emerging Fc-effector function that may aid in the control and clearance of HIV infection.

  20. Attachment Site Cysteine Thiol pKa Is a Key Driver for Site-Dependent Stability of THIOMAB Antibody-Drug Conjugates.

    Science.gov (United States)

    Vollmar, Breanna S; Wei, Binqing; Ohri, Rachana; Zhou, Jianhui; He, Jintang; Yu, Shang-Fan; Leipold, Douglas; Cosino, Ely; Yee, Sharon; Fourie-O'Donohue, Aimee; Li, Guangmin; Phillips, Gail L; Kozak, Katherine R; Kamath, Amrita; Xu, Keyang; Lee, Genee; Lazar, Greg A; Erickson, Hans K

    2017-10-18

    The incorporation of cysteines into antibodies by mutagenesis allows for the direct conjugation of small molecules to specific sites on the antibody via disulfide bonds. The stability of the disulfide bond linkage between the small molecule and the antibody is highly dependent on the location of the engineered cysteine in either the heavy chain (HC) or the light chain (LC) of the antibody. Here, we explore the basis for this site-dependent stability. We evaluated the in vivo efficacy and pharmacokinetics of five different cysteine mutants of trastuzumab conjugated to a pyrrolobenzodiazepine (PBD) via disulfide bonds. A significant correlation was observed between disulfide stability and efficacy for the conjugates. We hypothesized that the observed site-dependent stability of the disulfide-linked conjugates could be due to differences in the attachment site cysteine thiol pK a . We measured the cysteine thiol pK a using isothermal titration calorimetry (ITC) and found that the variants with the highest thiol pK a (LC K149C and HC A140C) were found to yield the conjugates with the greatest in vivo stability. Guided by homology modeling, we identified several mutations adjacent to LC K149C that reduced the cysteine thiol pK a and, thus, decreased the in vivo stability of the disulfide-linked PBD conjugated to LC K149C. We also present results suggesting that the high thiol pK a of LC K149C is responsible for the sustained circulation stability of LC K149C TDCs utilizing a maleimide-based linker. Taken together, our results provide evidence that the site-dependent stability of cys-engineered antibody-drug conjugates may be explained by interactions between the engineered cysteine and the local protein environment that serves to modulate the side-chain thiol pK a . The influence of cysteine thiol pK a on stability and efficacy offers a new parameter for the optimization of ADCs that utilize cysteine engineering.

  1. N-Acetylgalactosamino Dendrons as Clearing Agents to Enhance Liver Targeting of Model Antibody-Fusion Protein

    Science.gov (United States)

    Yoo, Barney; Cheal, Sarah M.; Torchon, Geralda; Dilhas, Anna; Yang, Guangbin; Pu, Jun; Punzalan, Blesida; Larson, Steven M.; Ouerfelli, Ouathek

    2014-01-01

    Dendrimer clearing agents represent a unique class of compounds for use in multistep targeting (MST) in radioimmunotherapy and imaging. These compounds were developed to facilitate the removal of excess tumor-targeting monoclonal antibody (mAb) prior to administration of the radionuclide to minimize exposure of normal tissue to radiation. Clearing agents are designed to capture the circulating mAb, and target it to the liver for metabolism. Glycodendrons are ideally suited for MST applications as these highly branched compounds are chemically well-defined thus advantageous over heterogeneous macromolecules. Previous studies have described glycodendron 3 as a clearing agent for use in three-step MST protocols, and early in vivo assessment of 3 showed promise. However, synthetic challenges have hampered its availability for further development. In this report we describe a new sequence of chemical steps which enables the straightforward synthesis and analytical characterization of this class of dendrons. With accessibility and analytical identification solved, we sought to evaluate both lower and higher generation dendrons for hepatocyte targeting as well as clearance of a model protein. We prepared a series of clearing agents where a single biotin is connected to glycodendrons displaying four, eight, sixteen or thirty-two α-thio-N-acetylgalactosamine (α–SGalNAc) units, resulting in compounds with molecular weights ranging from 2 to 17 kDa, respectively. These compounds were fully characterized by LCMS and NMR. We then evaluated the capacity of these agents to clear a model 131I-labeled single chain variable fragment antibody-streptavidin (131I-scFv-SAv) fusion protein from blood and tissue in mice, and compared their clearing efficiencies to that of a 500 kDa dextran-biotin conjugate. Glycodendrons and dextran-biotin exhibited enhanced blood clearance of the scFv-SAv construct. Biodistribution analysis showed liver targeting/uptake of the scFv-SAv construct to

  2. Antibodies to phosphatidylserine/prothrombin (aPS/PT) enhanced the diagnostic performance in Chinese patients with antiphospholipid syndrome.

    Science.gov (United States)

    Zhang, Shulan; Wu, Ziyan; Zhang, Wen; Zhao, Jiuliang; Norman, Gary L; Zeng, Xiaofeng; Zhang, Fengchun; Li, Yongzhe

    2018-03-21

    Increasing evidence has highlighted the role of non-criteria antiphospholipid antibodies (aPLs) as important supplements to the current criteria aPLs for the diagnosis of antiphospholipid syndrome (APS). In this retrospective study, we evaluated the clinical relevance of antibodies to phosphatidylserine/prothrombin (aPS/PT) in Chinese patients with APS. A total of 441 subjects were tested, including 101 patients with primary APS (PAPS), 140 patients with secondary APS (SAPS), 161 disease controls (DCs) and 39 healthy controls (HCs). Serum IgG/IgM aPS/PT was determined by ELISA. The levels of IgG/IgM aPS/PT were significantly increased in patients with APS compared with DCs and HCs. IgG and IgM aPS/PT were present in 29.7% and 54.5% of PAPS, and 42.1% and 53.6% of SAPS, respectively. For diagnosis of APS, IgG aCL exhibited the highest positive likelihood ratio (LR+) of 21.60, followed by LA (13.84), IgG aβ2GP1 (9.19) and IgG aPS/PT (8.49). aPS/PT was detected in 13.3% of seronegative PAPS patients and 31.3% of seronegative SAPS patients. LA exhibited the highest OR of 3.64 in identifying patients with thrombosis, followed by IgG aCL (OR, 2.63), IgG aPS/PT (OR, 2.55) and IgG aβ2GP1 (OR, 2.33). LA and IgG aCL were correlated with both arterial and venous thrombosis, whereas IgG aPS/PT and IgG aβ2GP1 correlated with venous or arterial thrombosis, respectively. Our findings suggest that the inclusion of IgG/IgM aPS/PT may enhance the diagnostic performance for APS, especially in those in whom APS is highly suspected, but conventional aPLs are repeatedly negative. In addition, IgG aPS/PT may contribute to identify patients at risk of thrombosis.

  3. How Recommender Systems in Technology-Enhanced Learning depend on Context

    NARCIS (Netherlands)

    Drachsler, Hendrik; Manouselis, Nikos

    2009-01-01

    Drachsler, H., & Manouselis, N. (2009). How Recommender Systems in Technology-Enhanced Learning depend on Context. Presentation given at the 1st workshop on Context-aware Recommender Systems for Learning at the Alpine Rendez-Vous 2009. November, 30 - December, 3, 2009, Garmisch-Patenkirchen,

  4. Prolonged local forearm hyperinsulinemia induces sustained enhancement of nitric oxide-dependent vasodilation in healthy subjects

    DEFF Research Database (Denmark)

    Hermann, Thomas S; Ihlemann, Nikolaj; Dominguez, Helena

    2005-01-01

    -dependent and -independent vasodilation.N(G)-monomethyl-L-arginine (L-NMMA) was coinfused to test the degree of nitric oxide (NO)-mediated vasodilation. Insulin infusion for 60 min enhanced serotonin-induced vasodilation by 37% compared to vehicle, p = .016. This increase was maintained for 4 h and was blocked by L...

  5. Combination of poly I:C and Pam3CSK4 enhances activation of B cells in vitro and boosts antibody responses to protein vaccines in vivo.

    Directory of Open Access Journals (Sweden)

    Genevieve M Weir

    Full Text Available Vaccines that can rapidly induce strong and robust antibody-mediated immunity could improve protection from certain infectious diseases for which current vaccine formulations are inefficient. For indications such as anthrax and influenza, antibody production in vivo is a correlate of efficacy. Toll-like receptor (TLR agonists are frequently studied for their role as vaccine adjuvants, largely because of their ability to enhance initiation of immune responses to antigens by activating dendritic cells. However, TLRs are also expressed on B cells and may contribute to effective B cell activation and promote differentiation into antigen-specific antibody producing plasma cells in vivo. We sought to discover an adjuvant system that could be used to augment antibody responses to influenza and anthrax vaccines. We first characterized an adjuvant system in vitro which consisted of two TLR ligands, poly I:C (TLR3 and Pam3CSK4 (TLR2, by evaluating its effects on B cell activation. Each agonist enhanced B cell activation through increased expression of surface receptors, cytokine secretion and proliferation. However, when B cells were stimulated with poly I:C and Pam3CSK4 in combination, further enhancement to cell activation was observed. Using B cells isolated from knockout mice we confirmed that poly I:C and Pam3CSK4 were signaling through TLR3 and TLR2, respectively. B cells activated with Poly I:C and Pam3CSK4 displayed enhanced capacity to stimulate allogeneic CD4+ T cell activation and differentiate into antibody-producing plasma cells in vitro. Mice vaccinated with influenza or anthrax antigens formulated with poly I:C and Pam3CSK4 in DepoVax™ vaccine platform developed a rapid and strong antigen-specific serum antibody titer that persisted for at least 12 weeks after a single immunization. These results demonstrate that combinations of TLR adjuvants promote more effective B cell activation in vitro and can be used to augment antibody responses

  6. Differential regulation of T-cell dependent and T-cell independent antibody responses through arginine methyltransferase PRMT1 in vivo.

    Science.gov (United States)

    Hata, Kikumi; Yanase, Noriko; Sudo, Katsuko; Kiyonari, Hiroshi; Mukumoto, Yoshiko; Mizuguchi, Junichiro; Yokosuka, Tadashi

    2016-04-01

    Protein arginine methyltransferase 1 (PRMT1), a major PRMT in mammalian cells, has been shown to play a crucial role in multiple biological functions in vitro. To explore the role of PRMT1 in B cells in vivo, we generated B cell-specific PRMT1-deficient (Prmt1(-/-) ) mice using a Cre-loxP system. Prmt1(-/-) mice showed a defect in B-cell development with diminished levels of serum antibodies. Antibody responses in Prmt1(-/-) mice were absent after stimulation with the type 2 T cell-independent antigen NP-Ficoll but intact after stimulation with the T cell-dependent antigen NP-OVA. Our findings comprise the first evidence showing that PRMT1 is necessary for lymphocyte functions in vivo. © 2016 Federation of European Biochemical Societies.

  7. Antibodies against glycolipids enhance antifungal activity of macrophages and reduce fungal burden after infection with Paracoccidioides brasiliensis

    Directory of Open Access Journals (Sweden)

    Renata Amelia eBueno

    2016-02-01

    Full Text Available Paracoccidioidomycosis is a fungal disease endemic in Latin America. Polyclonal antibodies to acidic glycosphingolipids (GSLs from Paracoccidioides brasiliensis opsonized yeast forms in vitro increasing phagocytosis and reduced the fungal burden of infected animals. Antibodies to GSL were active in both prophylactic and therapeutic protocols using a murine intratracheal infection model. Pathological examination of the lungs of animals treated with antibodies to GSL showed well-organized granulomas and minimally damaged parenchyma compared to the untreated control. Murine peritoneal macrophages activated by IFN-γ and incubated with antibodies against acidic GSLs more effectively phagocytosed and killed P. brasiliensis yeast cells as well as produced more nitric oxide compared to controls. The present work discloses a novel target of protective antibodies against P. brasiliensis adding to other well-studied mediators of the immune response to this fungus.

  8. DNA vaccine molecular adjuvants SP-D-BAFF and SP-D-APRIL enhance anti-gp120 immune response and increase HIV-1 neutralizing antibody titers.

    Science.gov (United States)

    Gupta, Sachin; Clark, Emily S; Termini, James M; Boucher, Justin; Kanagavelu, Saravana; LeBranche, Celia C; Abraham, Sakhi; Montefiori, David C; Khan, Wasif N; Stone, Geoffrey W

    2015-04-01

    Broadly neutralizing antibodies (bNAbs) specific for conserved epitopes on the HIV-1 envelope (Env) are believed to be essential for protection against multiple HIV-1 clades. However, vaccines capable of stimulating the production of bNAbs remain a major challenge. Given that polyreactivity and autoreactivity are considered important characteristics of anti-HIV bNAbs, we designed an HIV vaccine incorporating the molecular adjuvants BAFF (B cell activating factor) and APRIL (a proliferation-inducing ligand) with the potential to facilitate the maturation of polyreactive and autoreactive B cells as well as to enhance the affinity and/or avidity of Env-specific antibodies. We designed recombinant DNA plasmids encoding soluble multitrimers of BAFF and APRIL using surfactant protein D as a scaffold, and we vaccinated mice with these molecular adjuvants using DNA and DNA-protein vaccination strategies. We found that immunization of mice with a DNA vaccine encoding BAFF or APRIL multitrimers, together with interleukin 12 (IL-12) and membrane-bound HIV-1 Env gp140, induced neutralizing antibodies against tier 1 and tier 2 (vaccine strain) viruses. The APRIL-containing vaccine was particularly effective at generating tier 2 neutralizing antibodies following a protein boost. These BAFF and APRIL effects coincided with an enhanced germinal center (GC) reaction, increased anti-gp120 antibody-secreting cells, and increased anti-gp120 functional avidity. Notably, BAFF and APRIL did not cause indiscriminate B cell expansion or an increase in total IgG. We propose that BAFF and APRIL multitrimers are promising molecular adjuvants for vaccines designed to induce bNAbs against HIV-1. Recent identification of antibodies that neutralize most HIV-1 strains has revived hopes and efforts to create novel vaccines that can effectively stimulate HIV-1 neutralizing antibodies. However, the multiple immune evasion properties of HIV have hampered these efforts. These include the instability of

  9. Slaying the Trojan horse: natural killer cells exhibit robust anti-HIV-1 antibody-dependent activation and cytolysis against allogeneic T cells.

    Science.gov (United States)

    Gooneratne, Shayarana L; Richard, Jonathan; Lee, Wen Shi; Finzi, Andrés; Kent, Stephen J; Parsons, Matthew S

    2015-01-01

    Many attempts to design prophylactic human immunodeficiency virus type 1 (HIV-1) vaccines have focused on the induction of neutralizing antibodies (Abs) that block infection by free virions. Despite the focus on viral particles, virus-infected cells, which can be found within mucosal secretions, are more infectious than free virus both in vitro and in vivo. Furthermore, assessment of human transmission couples suggests infected seminal lymphocytes might be responsible for a proportion of HIV-1 transmissions. Although vaccines that induce neutralizing Abs are sought, only some broadly neutralizing Abs efficiently block cell-to-cell transmission of HIV-1. As HIV-1 vaccines need to elicit immune responses capable of controlling both free and cell-associated virus, we evaluated the potential of natural killer (NK) cells to respond in an Ab-dependent manner to allogeneic T cells bearing HIV-1 antigens. This study presents data measuring Ab-dependent anti-HIV-1 NK cell responses to primary and transformed allogeneic T-cell targets. We found that NK cells are robustly activated in an anti-HIV-1 Ab-dependent manner against allogeneic targets and that tested target cells are subject to Ab-dependent cytolysis. Furthermore, the educated KIR3DL1(+) NK cell subset from HLA-Bw4(+) individuals exhibits an activation advantage over the KIR3DL1(-) subset that contains both NK cells educated through other receptor/ligand combinations and uneducated NK cells. These results are intriguing and important for understanding the regulation of Ab-dependent NK cell responses and are potentially valuable for designing Ab-dependent therapies and/or vaccines. NK cell-mediated anti-HIV-1 antibody-dependent functions have been associated with protection from infection and disease progression; however, their role in protecting from infection with allogeneic cells infected with HIV-1 is unknown. We found that HIV-1-specific ADCC antibodies bound to allogeneic cells infected with HIV-1 or coated

  10. Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers

    Directory of Open Access Journals (Sweden)

    Rasool Suhail

    2007-09-01

    Full Text Available Abstract Background Amyloid-related degenerative diseases are associated with the accumulation of misfolded proteins as amyloid fibrils in tissue. In Alzheimer disease (AD, amyloid accumulates in several distinct types of insoluble plaque deposits, intracellular Aβ and as soluble oligomers and the relationships between these deposits and their pathological significance remains unclear. Conformation dependent antibodies have been reported that specifically recognize distinct assembly states of amyloids, including prefibrillar oligomers and fibrils. Results We immunized rabbits with a morphologically homogeneous population of Aβ42 fibrils. The resulting immune serum (OC specifically recognizes fibrils, but not random coil monomer or prefibrillar oligomers, indicating fibrils display a distinct conformation dependent epitope that is absent in prefibrillar oligomers. The fibril epitope is also displayed by fibrils of other types of amyloids, indicating that the epitope is a generic feature of the polypeptide backbone. The fibril specific antibody also recognizes 100,000 × G soluble fibrillar oligomers ranging in size from dimer to greater than 250 kDa on western blots. The fibrillar oligomers recognized by OC are immunologically distinct from prefibrillar oligomers recognized by A11, even though their sizes overlap broadly, indicating that size is not a reliable indicator of oligomer conformation. The immune response to prefibrillar oligomers and fibrils is not sequence specific and antisera of the same specificity are produced in response to immunization with islet amyloid polypeptide prefibrillar oligomer mimics and fibrils. The fibril specific antibodies stain all types of amyloid deposits in human AD brain. Diffuse amyloid deposits stain intensely with anti-fibril antibody although they are thioflavin S negative, suggesting that they are indeed fibrillar in conformation. OC also stains islet amyloid deposits in transgenic mouse models of type

  11. Epigenetic switch involved in activation of pioneer factor FOXA1-dependent enhancers

    Science.gov (United States)

    Sérandour, Aurélien A.; Avner, Stéphane; Percevault, Frédéric; Demay, Florence; Bizot, Maud; Lucchetti-Miganeh, Céline; Barloy-Hubler, Frédérique; Brown, Myles; Lupien, Mathieu; Métivier, Raphaël; Salbert, Gilles; Eeckhoute, Jérôme

    2011-01-01

    Transcription factors (TFs) bind specifically to discrete regions of mammalian genomes called cis-regulatory elements. Among those are enhancers, which play key roles in regulation of gene expression during development and differentiation. Despite the recognized central regulatory role exerted by chromatin in control of TF functions, much remains to be learned regarding the chromatin structure of enhancers and how it is established. Here, we have analyzed on a genomic-scale enhancers that recruit FOXA1, a pioneer transcription factor that triggers transcriptional competency of these cis-regulatory sites. Importantly, we found that FOXA1 binds to genomic regions showing local DNA hypomethylation and that its cell-type-specific recruitment to chromatin is linked to differential DNA methylation levels of its binding sites. Using neural differentiation as a model, we showed that induction of FOXA1 expression and its subsequent recruitment to enhancers is associated with DNA demethylation. Concomitantly, histone H3 lysine 4 methylation is induced at these enhancers. These epigenetic changes may both stabilize FOXA1 binding and allow for subsequent recruitment of transcriptional regulatory effectors. Interestingly, when cloned into reporter constructs, FOXA1-dependent enhancers were able to recapitulate their cell type specificity. However, their activities were inhibited by DNA methylation. Hence, these enhancers are intrinsic cell-type-specific regulatory regions of which activities have to be potentiated by FOXA1 through induction of an epigenetic switch that includes notably DNA demethylation. PMID:21233399

  12. Monoclonal Antibody against G Glycoprotein Increases Respiratory Syncytial Virus Clearance In Vivo and Prevents Vaccine-Enhanced Diseases.

    Directory of Open Access Journals (Sweden)

    Hyo-Jeong Lee

    Full Text Available Respiratory syncytial virus (RSV is a common cause of lower respiratory tract illness in infants, young children, and the elderly. The G glycoprotein plays a role in host cell attachment and also modulates the host immune response, thereby inducing disease pathogenesis. We generated two monoclonal antibodies (mAbs; 5H6 and 3A5 against G protein core fragment (Gcf, which consisted of amino acid residues 131 to 230 from RSV A2 G protein. Epitope mapping study revealed that 5H6 specifically binds to the G/164-176 peptide that includes conserved sequences shared by both RSV A and B subtypes, and 3A5 binds to the G/190-204 peptide. Studies with mutant Gcf proteins in which cysteine residues were substituted with alanine revealed that 5H6 requires four cysteines for binding and 3A5 binds to Gcf variants with alanine substitutions better than wild-type. To determine if these mAbs reduce pulmonary viral infection, BALB/c mice were administered mAb and subsequently challenged with RSV. On day 4 post-infection, lung viral titers were reduced by up to 93% with the 5H6 injection and 90% with the 3A5 injection, indicating that prophylactic injection of these mAbs contributes to RSV clearance in vivo. Importantly, 5H6 injection reduced vaccine-enhanced diseases. Overall, our results suggest that this novel anti-G mAb could be used as a prophylactic regimen against RSV diseases.

  13. Isotopic dependence of fusion enhancement of various heavy ion systems using energy dependent Woods-Saxon potential

    Science.gov (United States)

    Gautam, Manjeet Singh

    2015-01-01

    In the present work, the fusion of symmetric and asymmetric projectile-target combinations are deeply analyzed within the framework of energy dependent Woods-Saxon potential model (EDWSP model) in conjunction with one dimensional Wong formula and the coupled channel code CCFULL. The neutron transfer channels and the inelastic surface excitations of collision partners are dominating mode of couplings and the coupling of relative motion of colliding nuclei to such relevant internal degrees of freedom produces a significant fusion enhancement at sub-barrier energies. It is quite interesting that the effects of dominant intrinsic degrees of freedom such as multi-phonon vibrational states, neutron transfer channels and proton transfer channels can be simulated by introducing the energy dependence in the nucleus-nucleus potential (EDWSP model). In the EDWSP model calculations, a wide range of diffuseness parameter ranging from a = 0.85 fm to a = 0.97 fm, which is much larger than a value (a = 0.65 fm) extracted from the elastic scattering data, is needed to reproduce sub-barrier fusion data. However, such diffuseness anomaly, which might be an artifact of some dynamical effects, has been resolved by trajectory fluctuation dissipation (TFD) model wherein the resulting nucleus-nucleus potential possesses normal diffuseness parameter.

  14. Spectral dependence of the linewidth enhancement factor in quantum dot lasers

    Energy Technology Data Exchange (ETDEWEB)

    Zubov, F. I., E-mail: fedyazu@mail.ru [Russian Academy of Sciences, St. Petersburg Academic University-Nanotechnology Research and Education Center (Russian Federation); Shernyakov, Yu. M.; Maximov, M. V. [Russian Academy of Sciences, Ioffe Physical-Technical Institute (Russian Federation); Zhukov, A. E. [Russian Academy of Sciences, St. Petersburg Academic University-Nanotechnology Research and Education Center (Russian Federation); Livshits, D. A. [Innolume GmbH (Germany); Payusov, A. S.; Nadtochiy, A. M. [Russian Academy of Sciences, Ioffe Physical-Technical Institute (Russian Federation); Savelyev, A. V.; Kryzhanovskaya, N. V. [Russian Academy of Sciences, St. Petersburg Academic University-Nanotechnology Research and Education Center (Russian Federation); Gordeev, N. Yu. [Russian Academy of Sciences, Ioffe Physical-Technical Institute (Russian Federation)

    2013-12-15

    The spectral analysis of amplified spontaneous emission is used to determine the linewidth enhancement factor (α-factor) in lasers based on InAs/InGaAs quantum dots (QDs) in a wide spectral range near the ground-state optical transition energy. The effect of the pump current and number of QDs on the spectral dependences of the α-factor is examined. The temperature dependence of the spectra of the α-factor is experimentally determined for the first time for lasers with InAs/InGaAs QDs. An explanation is suggested for the observed anomalous decrease in the α-factor with increasing temperature.

  15. Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier

    International Nuclear Information System (INIS)

    Friden, P.M.; Walus, L.R.; Musso, G.F.; Taylor, M.A.; Malfroy, B.; Starzyk, R.M.

    1991-01-01

    Delivery of nonlipophilic drugs to the brain is hindered by the tightly apposed capillary endothelial cells that make up the blood-brain barrier. The authors have examined the ability of a monoclonal antibody (OX-26), which recognizes the rat transferrin receptor, to function as a carrier for the delivery of drugs across the blood-brain barrier. This antibody, which was previously shown to bind preferentially to capillary endothelial cells in the brain after intravenous administration, labels the entire cerebrovascular bed in a dose-dependent manner. The initially uniform labeling of brain capillaries becomes extremely punctate ∼ 4 hr after injection, suggesting a time-dependent sequestering of the antibody. Capillary-depletion experiments, in which the brain is separated into capillary and parenchymal fractions, show a time-dependent migration of radiolabeled antibody from the capillaries into the brain parenchyma, which is consistent with the transcytosis of compounds across the blood-brain barrier. Antibody-methotrexate conjugates were tested in vivo to assess the carrier ability of this antibody. Immunohistochemical staining for either component of an OX-26-methotrexate conjugate revealed patterns of cerebrovascular labeling identical to those observed with the unaltered antibody. Accumulation of radiolabeled methotrexate in the brain parenchyma is greatly enhanced when the drug is conjugated to OX-26

  16. Enhanced diffusion of oxygen depending on Fermi level position in heavily boron-doped silicon

    Energy Technology Data Exchange (ETDEWEB)

    Torigoe, Kazuhisa, E-mail: ktorigoe@sumcosi.com; Fujise, Jun; Ono, Toshiaki [Technology Division, Advanced Evaluation and Technology Development Department, SUMCO Corporation, 1-52 Kubara, Yamashiro-cho, Imari, Saga 849-4256 (Japan); Nakamura, Kozo [Department of Communication Engineering, Okayama Prefectural University, 111 Kuboki, Soja, Okayama 719-1197 (Japan)

    2014-11-21

    The enhanced diffusivity of oxygen in heavily boron doped silicon was obtained by analyzing oxygen out-diffusion profile changes found at the interface between a lightly boron-doped silicon epitaxial layer and a heavily boron-doped silicon substrate by secondary ion mass spectrometry. It was found that the diffusivity is proportional to the square root of boron concentration in the range of 10{sup 18 }cm{sup −3}–10{sup 19 }cm{sup −3} at temperatures from 750 °C to 950 °C. The model based on the diffusion of oxygen dimers in double positive charge state could explain the enhanced diffusion. We have concluded that oxygen diffusion enhanced in heavily boron-doped silicon is attributed to oxygen dimers ionized depending on Fermi level position.

  17. Enhanced performance of an innovative dengue IgG/IgM rapid diagnostic test using an anti-dengue EDI monoclonal antibody and dengue virus antigen

    Science.gov (United States)

    Lee, Jihoo; Kim, Young-Eun; Kim, Hak-Yong; Sinniah, Mangalam; Chong, Chom-Kyu; Song, Hyun-Ok

    2015-01-01

    High levels of anti-dengue IgM or IgG can be detected using numerous rapid diagnostic tests (RDTs). However, the sensitivity and specificity of these tests are reduced by changes in envelope glycoprotein antigenicity that inevitably occur in limited expression systems. A novel RDT was designed to enhance diagnostic sensitivity. Dengue viruses cultured in animal cells were used as antigens to retain the native viral coat protein. Monoclonal antibodies (mAbs) were then developed, for the first time, against domain I of envelope glycoprotein (EDI). The anti-dengue EDI mAb was employed as a capturer, and EDII and EDIII, which are mainly involved in the induction of neutralizing antibodies in patients, were fully available to bind to anti-dengue IgM or IgG in patients. A one-way automatic blood separation device prevented reverse migration of plasma and maximize the capture of anti-dengue antibodies at the test lines. A clinical evaluation in the field proved that the novel RDT (sensitivities of 96.5% and 96.7% for anti-dengue IgM and IgG) is more effective in detecting anti-dengue antibodies than two major commercial tests (sensitivities of 54.8% and 82% for SD BIOLINE; 50.4% and 75.3% for PanBio). The innovative format of RDT can be applied to other infectious viral diseases. PMID:26655854

  18. Microneedle-mediated immunization of an adenovirus-based malaria vaccine enhances antigen-specific antibody immunity and reduces anti-vector responses compared to the intradermal route

    Science.gov (United States)

    Carey, John B.; Vrdoljak, Anto; O'Mahony, Conor; Hill, Adrian V. S.; Draper, Simon J.; Moore, Anne C.

    2014-01-01

    Substantial effort has been placed in developing efficacious recombinant attenuated adenovirus-based vaccines. However induction of immunity to the vector is a significant obstacle to its repeated use. Here we demonstrate that skin-based delivery of an adenovirus-based malaria vaccine, HAdV5-PyMSP142, to mice using silicon microneedles induces equivalent or enhanced antibody responses to the encoded antigen, however it results in decreased anti-vector responses, compared to intradermal delivery. Microneedle-mediated vaccine priming and resultant induction of low anti-vector antibody titres permitted repeated use of the same adenovirus vaccine vector. This resulted in significantly increased antigen-specific antibody responses in these mice compared to ID-treated mice. Boosting with a heterologous vaccine; MVA-PyMSP142 also resulted in significantly greater antibody responses in mice primed with HAdV5-PyMSP142 using MN compared to the ID route. The highest protection against blood-stage malaria challenge was observed when a heterologous route of immunization (MN/ID) was used. Therefore, microneedle-mediated immunization has potential to both overcome some of the logistic obstacles surrounding needle-and-syringe-based immunization as well as to facilitate the repeated use of the same adenovirus vaccine thereby potentially reducing manufacturing costs of multiple vaccines. This could have important benefits in the clinical ease of use of adenovirus-based immunization strategies. PMID:25142082

  19. Enhanced size-dependent piezoelectricity and elasticity in nanostructures due to the flexoelectric effect

    Science.gov (United States)

    Majdoub, M. S.; Sharma, P.; Cagin, T.

    2008-03-01

    Crystalline piezoelectric dielectrics electrically polarize upon application of uniform mechanical strain. Inhomogeneous strain, however, locally breaks inversion symmetry and can potentially polarize even nonpiezoelectric (centrosymmetric) dielectrics. Flexoelectricity—the coupling of strain gradient to polarization—is expected to show a strong size dependency due to the scaling of strain gradients with structural feature size. In this study, using a combination of atomistic and theoretical approaches, we investigate the “effective” size-dependent piezoelectric and elastic behavior of inhomogeneously strained nonpiezoelectric and piezoelectric nanostructures. In particular, to obtain analytical results and tease out physical insights, we analyze a paradigmatic nanoscale cantilever beam. We find that in materials that are intrinsically piezoelectric, the flexoelectricity and piezoelectricity effects do not add linearly and exhibit a nonlinear interaction. The latter leads to a strong size-dependent enhancement of the apparent piezoelectric coefficient resulting in, for example, a “giant” 500% enhancement over bulk properties in BaTiO3 for a beam thickness of 5nm . Correspondingly, for nonpiezoelectric materials also, the enhancement is nontrivial (e.g., 80% for 5nm size in paraelectric BaTiO3 phase). Flexoelectricity also modifies the apparent elastic modulus of nanostructures, exhibiting an asymptotic scaling of 1/h2 , where h is the characteristic feature size. Our major predictions are verified by quantum mechanically derived force-field-based molecular dynamics for two phases (cubic and tetragonal) of BaTiO3 .

  20. Lack of reinforcement enhancing effects of nicotine in non-dependent smokers.

    Science.gov (United States)

    Perkins, Kenneth A; Grottenthaler, Amy; Wilson, Annette S

    2009-09-01

    Recent animal research has shown that, aside from its primary and secondary reinforcing effects, nicotine may enhance reinforcement from stimuli unrelated to nicotine intake. Little human research has directly examined this potentially important influence of nicotine. We report two virtually identical studies examining the influence of nicotine, via nasal spray (study 1) and cigarettes (study 2), on the reinforcing effects of rewards unrelated to nicotine intake. Both studies involved young adults with some past smoking exposure but no history of nicotine dependence. Reinforcement was assessed by responses on a simple operant computer task reinforced by: money, music, the termination of aversive noise, or no reward (control). Participants responded for rewards on three separate sessions, involving intermittent dosing of 0, 5, or 10 microg/kg nicotine via nasal spray (study 1) or the smoking of 0.05 or 0.6 mg nicotine cigarettes or no smoking (study 2). Results showed no effects of nicotine, by nasal spray or cigarette smoking, on reinforced responses, although nicotine increased some subjective responses (e.g. head rush/buzzed, liking). Nicotine via smoking also did not influence affect or hedonic ratings of slides varying in mood valence in an exploratory trial in study 2. These results do not support the notion that nicotine per se enhances the reinforcing value of other reinforcers in humans. Any reinforcement enhancing effects of nicotine in humans may be specific to dependent smokers or may be relatively narrow and dependent upon procedural conditions different from those in the current studies.

  1. The diagnostic sensitivity of dengue rapid test assays is significantly enhanced by using a combined antigen and antibody testing approach.

    Directory of Open Access Journals (Sweden)

    Scott R Fry

    2011-06-01

    Full Text Available BACKGROUND: Serological tests for IgM and IgG are routinely used in clinical laboratories for the rapid diagnosis of dengue and can differentiate between primary and secondary infections. Dengue virus non-structural protein 1 (NS1 has been identified as an early marker for acute dengue, and is typically present between days 1-9 post-onset of illness but following seroconversion it can be difficult to detect in serum. AIMS: To evaluate the performance of a newly developed Panbio® Dengue Early Rapid test for NS1 and determine if it can improve diagnostic sensitivity when used in combination with a commercial IgM/IgG rapid test. METHODOLOGY: The clinical performance of the Dengue Early Rapid was evaluated in a retrospective study in Vietnam with 198 acute laboratory-confirmed positive and 100 negative samples. The performance of the Dengue Early Rapid in combination with the IgM/IgG Rapid test was also evaluated in Malaysia with 263 laboratory-confirmed positive and 30 negative samples. KEY RESULTS: In Vietnam the sensitivity and specificity of the test was 69.2% (95% CI: 62.8% to 75.6% and 96% (95% CI: 92.2% to 99.8 respectively. In Malaysia the performance was similar with 68.9% sensitivity (95% CI: 61.8% to 76.1% and 96.7% specificity (95% CI: 82.8% to 99.9% compared to RT-PCR. Importantly, when the Dengue Early Rapid test was used in combination with the IgM/IgG test the sensitivity increased to 93.0%. When the two tests were compared at each day post-onset of illness there was clear differentiation between the antigen and antibody markers. CONCLUSIONS: This study highlights that using dengue NS1 antigen detection in combination with anti-glycoprotein E IgM and IgG serology can significantly increase the sensitivity of acute dengue diagnosis and extends the possible window of detection to include very early acute samples and enhances the clinical utility of rapid immunochromatographic testing for dengue.

  2. λ Light Chain Bias Associated With Enhanced Binding and Function of Anti-HIV Env Glycoprotein Antibodies.

    Science.gov (United States)

    Sajadi, Mohammad M; Farshidpour, Maham; Brown, Eric P; Ouyang, Xin; Seaman, Michael S; Pazgier, Marzena; Ackerman, Margaret E; Robinson, Harriet; Tomaras, Georgia; Parsons, Matthew S; Charurat, Manhattan; DeVico, Anthony L; Redfield, Robert R; Lewis, George K

    2016-01-01

    The humoral response to human immunodeficiency virus (HIV) remains incompletely understood. In this report, we describe biased λ light chain use during the HIV Env glycoprotein (Env) response in HIV infection and vaccination. We examined HIV Env binding (and neutralization) in the context of light chain use in subjects with acute HIV infection, chronic HIV infection, and among HIV vaccinees. In all populations tested, there was a λ chain bias for HIV Env binding antibodies, compared with other HIV antigens (such as p24) or tetanus toxoid. In subjects with chronic HIV infection, a λ bias was noted for neutralization, with λ antibodies accounting for up to 90% of all neutralization activity observed. This is the first report of antibody function in a human infection being tied to light chain use. In HIV infection, antibodies expressing λ light chains tended to have longer CDRL3s, increased light chain contact with HIV Env, and less hypermutation in the heavy chain, compared with antibodies using the κ light chain. These data also support an evolutionary model for the understanding the various κ to λ light chain ratios observed across species and suggest that the λ light chain bias against HIV provides the host an advantage in developing a more efficient humoral response. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  3. Targeted Delivery of Neutralizing Anti-C5 Antibody to Renal Endothelium Prevents Complement-Dependent Tissue Damage

    Directory of Open Access Journals (Sweden)

    Paolo Durigutto

    2017-09-01

    Full Text Available Complement activation is largely implicated in the pathogenesis of several clinical conditions and its therapeutic neutralization has proven effective in preventing tissue and organ damage. A problem that still needs to be solved in the therapeutic control of complement-mediated diseases is how to avoid side effects associated with chronic neutralization of the complement system, in particular, the increased risk of infections. We addressed this issue developing a strategy based on the preferential delivery of a C5 complement inhibitor to the organ involved in the pathologic process. To this end, we generated Ergidina, a neutralizing recombinant anti-C5 human antibody coupled with a cyclic-RGD peptide, with a distinctive homing property for ischemic endothelial cells and effective in controlling tissue damage in a rat model of renal ischemia/reperfusion injury (IRI. As a result of its preferential localization on renal endothelium, the molecule induced complete inhibition of complement activation at tissue level, and local protection from complement-mediated tissue damage without affecting circulating C5. The ex vivo binding of Ergidina to surgically removed kidney exposed to cold ischemia supports its therapeutic use to prevent posttransplant IRI leading to delay of graft function. Moreover, the finding that the ex vivo binding of Ergidina was not restricted to the kidney, but was also seen on ischemic heart, suggests that this RGD-targeted anti-C5 antibody may represent a useful tool to treat organs prior to transplantation. Based on this evidence, we propose preliminary data showing that Ergidina is a novel targeted drug to prevent complement activation on the endothelium of ischemic kidney.

  4. YAP/TAZ enhance mammalian embryonic neural stem cell characteristics in a Tead-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Han, Dasol; Byun, Sung-Hyun; Park, Soojeong; Kim, Juwan; Kim, Inhee; Ha, Soobong; Kwon, Mookwang; Yoon, Keejung, E-mail: keejung@skku.edu

    2015-02-27

    Mammalian brain development is regulated by multiple signaling pathways controlling cell proliferation, migration and differentiation. Here we show that YAP/TAZ enhance embryonic neural stem cell characteristics in a cell autonomous fashion using diverse experimental approaches. Introduction of retroviral vectors expressing YAP or TAZ into the mouse embryonic brain induced cell localization in the ventricular zone (VZ), which is the embryonic neural stem cell niche. This change in cell distribution in the cortical layer is due to the increased stemness of infected cells; YAP-expressing cells were colabeled with Sox2, a neural stem cell marker, and YAP/TAZ increased the frequency and size of neurospheres, indicating enhanced self-renewal- and proliferative ability of neural stem cells. These effects appear to be TEA domain family transcription factor (Tead)–dependent; a Tead binding-defective YAP mutant lost the ability to promote neural stem cell characteristics. Consistently, in utero gene transfer of a constitutively active form of Tead2 (Tead2-VP16) recapitulated all the features of YAP/TAZ overexpression, and dominant negative Tead2-EnR resulted in marked cell exit from the VZ toward outer cortical layers. Taken together, these results indicate that the Tead-dependent YAP/TAZ signaling pathway plays important roles in neural stem cell maintenance by enhancing stemness of neural stem cells during mammalian brain development. - Highlights: • Roles of YAP and Tead in vivo during mammalian brain development are clarified. • Expression of YAP promotes embryonic neural stem cell characteristics in vivo in a cell autonomous fashion. • Enhancement of neural stem cell characteristics by YAP depends on Tead. • Transcriptionally active form of Tead alone can recapitulate the effects of YAP. • Transcriptionally repressive form of Tead severely reduces stem cell characteristics.

  5. Measured Wavelength-Dependent Absorption Enhancement of Internally Mixed Black Carbon with Absorbing and Nonabsorbing Materials.

    Science.gov (United States)

    You, Rian; Radney, James G; Zachariah, Michael R; Zangmeister, Christopher D

    2016-08-02

    Optical absorption spectra of laboratory generated aerosols consisting of black carbon (BC) internally mixed with nonabsorbing materials (ammonium sulfate, AS, and sodium chloride, NaCl) and BC with a weakly absorbing brown carbon surrogate derived from humic acid (HA) were measured across the visible to near-IR (550 to 840 nm). Spectra were measured in situ using a photoacoustic spectrometer and step-scanning a supercontinuum laser source with a tunable wavelength and bandwidth filter. BC had a mass-specific absorption cross section (MAC) of 7.89 ± 0.25 m(2) g(-1) at λ = 550 nm and an absorption Ångström exponent (AAE) of 1.03 ± 0.09 (2σ). For internally mixed BC, the ratio of BC mass to the total mass of the mixture was chosen as 0.13 to mimic particles observed in the terrestrial atmosphere. The manner in which BC mixed with each material was determined from transmission electron microscopy (TEM). AS/BC and HA/BC particles were fully internally mixed, and the BC was both internally and externally mixed for NaCl/BC particles. The AS/BC, NaCl/BC, and HA/BC particles had AAEs of 1.43 ± 0.05, 1.34 ± 0.06, and 1.91 ± 0.05, respectively. The observed absorption enhancement of mixed BC relative to the pure BC was wavelength dependent for AS/BC and decreased from 1.5 at λ = 550 nm with increasing wavelength while the NaCl/BC enhancement was essentially wavelength independent. For HA/BC, the enhancement ranged from 2 to 3 and was strongly wavelength dependent. Removal of the HA absorption contribution to enhancement revealed that the enhancement was ≈1.5 and independent of wavelength.

  6. Conglutinin exhibits a complement-dependent enhancement of the respiratory burst of phagocytes stimulated by E. coli

    DEFF Research Database (Denmark)

    Friis, P; Svehag, S E; Andersen, Ove

    1991-01-01

    . Conglutinin enhances, in a dose-dependent manner, the respiratory burst of spleen cells stimulated with serum-opsonized Escherichia coli. The enhancement was only demonstrable in the presence of a functional complement system. The conglutinin-mediated enhancement of the respiratory burst was inhibited...

  7. Complement receptor mediates enhanced flavivirus replication in macrophages

    OpenAIRE

    1983-01-01

    Evidence is presented that M phi complement receptors (CR3) mediate IgM- dependent enhancement of flavivirus replication in the presence of complement. Enhancement is blocked by pretreatment of macrophages with monoclonal antibody Ml/70, which inhibits CR3 binding, but not by pretreatment with monoclonal antibody 2.4G2, which inhibits FcR binding.

  8. ICAM-1-based rabies virus vaccine shows increased infection and activation of primary murine B cells in vitro and enhanced antibody titers in-vivo.

    Science.gov (United States)

    Norton, James E; Lytle, Andrew G; Shen, Shixue; Tzvetkov, Evgeni P; Dorfmeier, Corin L; McGettigan, James P

    2014-01-01

    We have previously shown that live-attenuated rabies virus (RABV)-based vaccines infect and directly activate murine and human primary B cells in-vitro, which we propose can be exploited to help develop a single-dose RABV-based vaccine. Here we report on a novel approach to utilize the binding of Intracellular Adhesion Molecule-1 (ICAM-1) to its binding partner, Lymphocyte Function-associated Antigen-1 (LFA-1), on B cells to enhance B cell activation and RABV-specific antibody responses. We used a reverse genetics approach to clone, recover, and characterize a live-attenuated recombinant RABV-based vaccine expressing the murine Icam1 gene (rRABV-mICAM-1). We show that the murine ICAM-1 gene product is incorporated into virus particles, potentially exposing ICAM-1 to extracellular binding partners. While rRABV-mICAM-1 showed 10-100-fold decrease in viral titers on baby hamster kidney cells compared to the parental virus (rRABV), rRABV-mICAM-1 infected and activated primary murine B cells in-vitro more efficiently than rRABV, as indicated by significant upregulation of CD69, CD40, and MHCII on the surface of infected B cells. ICAM-1 expression on the virus surface was responsible for enhanced B cell infection since pre-treating rRABV-mICAM-1 with a neutralizing anti-ICAM-1 antibody reduced B cell infection to levels observed with rRABV alone. Furthermore, 100-fold less rRABV-mICAM-1 was needed to induce antibody titers in immunized mice equivalent to antibody titers observed in rRABV-immunized mice. Of note, only 10(3) focus forming units (ffu)/mouse of rRABV-mICAM-1 was needed to induce significant anti-RABV antibody titers as early as five days post-immunization. As both speed and potency of antibody responses are important in controlling human RABV infection in a post-exposure setting, these data show that expression of Icam1 from the RABV genome, which is then incorporated into the virus particle, is a promising strategy for the development of a single-dose RABV

  9. ICAM-1-based rabies virus vaccine shows increased infection and activation of primary murine B cells in vitro and enhanced antibody titers in-vivo.

    Directory of Open Access Journals (Sweden)

    James E Norton

    Full Text Available We have previously shown that live-attenuated rabies virus (RABV-based vaccines infect and directly activate murine and human primary B cells in-vitro, which we propose can be exploited to help develop a single-dose RABV-based vaccine. Here we report on a novel approach to utilize the binding of Intracellular Adhesion Molecule-1 (ICAM-1 to its binding partner, Lymphocyte Function-associated Antigen-1 (LFA-1, on B cells to enhance B cell activation and RABV-specific antibody responses. We used a reverse genetics approach to clone, recover, and characterize a live-attenuated recombinant RABV-based vaccine expressing the murine Icam1 gene (rRABV-mICAM-1. We show that the murine ICAM-1 gene product is incorporated into virus particles, potentially exposing ICAM-1 to extracellular binding partners. While rRABV-mICAM-1 showed 10-100-fold decrease in viral titers on baby hamster kidney cells compared to the parental virus (rRABV, rRABV-mICAM-1 infected and activated primary murine B cells in-vitro more efficiently than rRABV, as indicated by significant upregulation of CD69, CD40, and MHCII on the surface of infected B cells. ICAM-1 expression on the virus surface was responsible for enhanced B cell infection since pre-treating rRABV-mICAM-1 with a neutralizing anti-ICAM-1 antibody reduced B cell infection to levels observed with rRABV alone. Furthermore, 100-fold less rRABV-mICAM-1 was needed to induce antibody titers in immunized mice equivalent to antibody titers observed in rRABV-immunized mice. Of note, only 10(3 focus forming units (ffu/mouse of rRABV-mICAM-1 was needed to induce significant anti-RABV antibody titers as early as five days post-immunization. As both speed and potency of antibody responses are important in controlling human RABV infection in a post-exposure setting, these data show that expression of Icam1 from the RABV genome, which is then incorporated into the virus particle, is a promising strategy for the development of a

  10. Macrophages are critical effectors of antibody therapies for cancer.

    Science.gov (United States)

    Weiskopf, Kipp; Weissman, Irving L

    2015-01-01

    Macrophages are innate immune cells that derive from circulating monocytes, reside in all tissues, and participate in many states of pathology. Macrophages play a dichotomous role in cancer, where they promote tumor growth but also serve as critical immune effectors of therapeutic antibodies. Macrophages express all classes of Fcγ receptors, and they have immense potential to destroy tumors via the process of antibody-dependent phagocytosis. A number of studies have demonstrated that macrophage phagocytosis is a major mechanism of action of many antibodies approved to treat cancer. Consequently, a number of approaches to augment macrophage responses to therapeutic antibodies are under investigation, including the exploration of new targets and development of antibodies with enhanced functions. For example, the interaction of CD47 with signal-regulatory protein α (SIRPα) serves as a myeloid-specific immune checkpoint that limits the response of macrophages to antibody therapies, and CD47-blocking agents overcome this barrier to augment phagocytosis. The response of macrophages to antibody therapies can also be enhanced with engineered Fc variants, bispecific antibodies, or antibody-drug conjugates. Macrophages have demonstrated success as effectors of cancer immunotherapy, and further investigation will unlock their full potential for the benefit of patients.

  11. YAP/TAZ enhance mammalian embryonic neural stem cell characteristics in a Tead-dependent manner.

    Science.gov (United States)

    Han, Dasol; Byun, Sung-Hyun; Park, Soojeong; Kim, Juwan; Kim, Inhee; Ha, Soobong; Kwon, Mookwang; Yoon, Keejung

    2015-02-27

    Mammalian brain development is regulated by multiple signaling pathways controlling cell proliferation, migration and differentiation. Here we show that YAP/TAZ enhance embryonic neural stem cell characteristics in a cell autonomous fashion using diverse experimental approaches. Introduction of retroviral vectors expressing YAP or TAZ into the mouse embryonic brain induced cell localization in the ventricular zone (VZ), which is the embryonic neural stem cell niche. This change in cell distribution in the cortical layer is due to the increased stemness of infected cells; YAP-expressing cells were colabeled with Sox2, a neural stem cell marker, and YAP/TAZ increased the frequency and size of neurospheres, indicating enhanced self-renewal- and proliferative ability of neural stem cells. These effects appear to be TEA domain family transcription factor (Tead)-dependent; a Tead binding-defective YAP mutant lost the ability to promote neural stem cell characteristics. Consistently, in utero gene transfer of a constitutively active form of Tead2 (Tead2-VP16) recapitulated all the features of YAP/TAZ overexpression, and dominant negative Tead2-EnR resulted in marked cell exit from the VZ toward outer cortical layers. Taken together, these results indicate that the Tead-dependent YAP/TAZ signaling pathway plays important roles in neural stem cell maintenance by enhancing stemness of neural stem cells during mammalian brain development. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Temperature dependence of CO2-enhanced primary production in the European Arctic Ocean

    KAUST Repository

    Holding, J. M.

    2015-08-31

    The Arctic Ocean is warming at two to three times the global rate1 and is perceived to be a bellwether for ocean acidification2, 3. Increased CO2 concentrations are expected to have a fertilization effect on marine autotrophs4, and higher temperatures should lead to increased rates of planktonic primary production5. Yet, simultaneous assessment of warming and increased CO2 on primary production in the Arctic has not been conducted. Here we test the expectation that CO2-enhanced gross primary production (GPP) may be temperature dependent, using data from several oceanographic cruises and experiments from both spring and summer in the European sector of the Arctic Ocean. Results confirm that CO2 enhances GPP (by a factor of up to ten) over a range of 145–2,099 μatm; however, the greatest effects are observed only at lower temperatures and are constrained by nutrient and light availability to the spring period. The temperature dependence of CO2-enhanced primary production has significant implications for metabolic balance in a warmer, CO2-enriched Arctic Ocean in the future. In particular, it indicates that a twofold increase in primary production during the spring is likely in the Arctic.

  13. Env-glycoprotein heterogeneity as a source of apparent synergy and enhanced cooperativity in inhibition of HIV-1 infection by neutralizing antibodies and entry inhibitors

    Science.gov (United States)

    Ketas, Thomas J.; Holuigue, Sophie; Matthews, Katie; Moore, John P.

    2011-01-01

    We measured the inhibition of infectivity of HIV-1 isolates and derivative clones by combinations of neutralizing antibodies (NAbs) and other entry inhibitors in a single-cycle-replication assay. Synergy was analyzed both by the current linear and a new nonlinear method. The new method reduced spurious indications of synergy and antagonism. Synergy between NAbs was overall weaker than between other entry inhibitors, and no stronger where one ligand is known to enhance the binding of another. However, synergy was stronger for a genetically heterogeneous HIV-1 R5 isolate than for its derivative clones. Enhanced cooperativity in inhibition by combinations, compared with individual inhibitors, correlated with increased synergy at higher levels of inhibition, while being less variable. Again, cooperativity enhancement was stronger for isolates than clones. We hypothesize that genetic, post-translational or conformational heterogeneity of the Env protein and of other targets for inhibitors can yield apparent synergy and increased cooperativity between inhibitors. PMID:22018634

  14. Phosphatidylserine-dependent anti-prothrombin antibodies (aPS/PT) in infliximab-treated patients with inflammatory bowel diseases.

    Science.gov (United States)

    Malíčková, Karin; Ďuricová, Dana; Bortlík, Martin; Janatková, Ivana; Zima, Tomáš; Lukáš, Milan

    2013-04-01

    To (1) examine the occurrence and concentrations of aPS/PT and aPL in inflammatory bowel disease (IBD) patients at the beginning of and during anti-TNF-alpha therapy with infliximab; (2) investigate the link of the aPS/PT and aPL presence with antibodies to infliximab (ATI) formation; and (3) examine possible clinical consequences of aPS/PT and/or aPL positivity in IBD patients. Thirty (30) IBD patients treated with infliximab were analyzed regarding aPS/PT, aPL, and ATI antibody serum levels by standardized ELISAs at treatment weeks 2 (W2) and 14 (W14). At W2, 40 % of infliximab-treated patients had elevated aPS/PT and 16.7 % had elevated aPL serum levels. At W14, the proportion of aPS/PT-positive sera decreased to 16.6 %, whereas aPL distribution remained unchanged. Moreover, concentrations of aPS/PT have shown significant differences at W2 (16.64 [10.06; 33.06] U for IgG and 18.46 [9.18; 32.48] U for IgM) and at W14 (8.24 [2.78; 19.82] U for IgG and 8.57 [5.55; 26.82] U for IgM), p = 0.009 and p = 0.003, respectively. In ATI-positive samples, aPS/PT IgG were more frequent (p = 0.001 for W2 and p = 0.003 for W14), whereas aPS/PT IgM and aPL IgG/IgM did not show such association. Higher concentrations of aPS/PT IgG and IgM were found in IBD patients at the beginning of the biological treatment period compared to the maintenance treatment period. Moreover, aPS/PT IgG were more frequent in ATI-positive individuals, which was not observed in aPL. We speculate that there is a relationship between the aPS/PT and the severity of inflammation and auto-aggressive processes in IBD.

  15. Guanfacine enhances inhibitory control and attentional shifting in early abstinent cocaine-dependent individuals.

    Science.gov (United States)

    Fox, Helen; Sofuoglu, Mehmet; Sinha, Rajita

    2015-03-01

    Attenuation of adrenergic drive and cognitive enhancement, via stimulation of alpha2 pre- and post-synaptic receptors, may selectively enhance executive performance in early abstinent cocaine-dependent individuals. As these cognitive processes underpin important treatment-related behaviors, the alpha2 agonist, guanfacine HCl, may represent an effective pharmaco-therapeutic intervention. Twenty-five early abstinent cocaine-dependent individuals were administered a battery of neurocognitive tasks on entry into treatment (baseline) and again following 3 weeks of either placebo or guanfacine treatment (up to 3 mg). Tasks included: Stop Signal, Stroop, 3-Dimentional Intra-dimensional/Extra-dimensional (IDED) task, Spatial Working Memory (SWM), Paired Associates Learning (PAL), Verbal Fluency and the Rey Auditory Verbal Learning Test (RAVLT). Compared with placebo, the guanfacine group demonstrated attenuated anxiety and negative affect as well as improved performance on selective executive tests. This included fewer directional errors on the stop signal task, fewer errors on the extra-dimensional shift component of the IDED task and better attentional switching during verbal fluency. Guanfacine did not improve strategic working memory or peripheral memory. Guanfacine improves selective cognitive processes which may underlie salient treatment-related regulatory behaviors. Alpha2 agonists may therefore represent important agents for cocaine dependence. © The Author(s) 2015.

  16. Hidden Lineage Complexity of Glycan-Dependent HIV-1 Broadly Neutralizing Antibodies Uncovered by Digital Panning and Native-Like gp140 Trimer

    Directory of Open Access Journals (Sweden)

    Linling He

    2017-08-01

    Full Text Available Germline precursors and intermediates of broadly neutralizing antibodies (bNAbs are essential to the understanding of humoral response to HIV-1 infection and B-cell lineage vaccine design. Using a native-like gp140 trimer probe, we examined antibody libraries constructed from donor-17, the source of glycan-dependent PGT121-class bNAbs recognizing the N332 supersite on the HIV-1 envelope glycoprotein. To facilitate this analysis, a digital panning method was devised that combines biopanning of phage-displayed antibody libraries, 900 bp long-read next-generation sequencing, and heavy/light (H/L-paired antibodyomics. In addition to single-chain variable fragments resembling the wild-type bNAbs, digital panning identified variants of PGT124 (a member of the PGT121 class with a unique insertion in the heavy chain complementarity-determining region 1, as well as intermediates of PGT124 exhibiting notable affinity for the native-like trimer and broad HIV-1 neutralization. In a competition assay, these bNAb intermediates could effectively compete with mouse sera induced by a scaffolded BG505 gp140.681 trimer for the N332 supersite. Our study thus reveals previously unrecognized lineage complexity of the PGT121-class bNAbs and provides an array of library-derived bNAb intermediates for evaluation of immunogens containing the N332 supersite. Digital panning may prove to be a valuable tool in future studies of bNAb diversity and lineage development.

  17. The impact of HLA class I-specific killer cell immunoglobulin-like receptors on antibody-dependent natural killer cell-mediated cytotoxicity and organ allograft rejection

    Directory of Open Access Journals (Sweden)

    Raja Rajalingam

    2016-12-01

    Full Text Available Natural killer (NK cells of the innate immune system are cytotoxic lymphocytes that play important roles following transplantation of solid organs and hematopoietic stem cells. Recognition of self HLA class I molecules by inhibitory killer cell immunoglobulin-like receptors (KIR is involved in the calibration of NK cell effector capacities during a developmental stage, allowing the subsequent recognition and elimination of target cells with decreased expression of self HLA class I (due to virus infection or tumor transformation or HLA class I disparities (in the setting of allogeneic transplantation. NK cells expressing an inhibitory KIR binding self HLA can be activated when confronted with allografts lacking a ligand for the inhibitory receptor. Following the response of the adaptive immune system, NK cells can further destroy allograft endothelium by antibody-dependent cell-mediated cytotoxicity (ADCC, triggered through cross-linking of the CD16 Fc receptor by donor-specific antibodies bound to allograft. Upon recognizing allogeneic target cells, NK cells also secrete cytokines and chemokines that drive maturation of dendritic cells to promote cellular and humoral adaptive immune responses against the allograft. The cumulative activating and inhibitory signals generated by ligation of the receptors regulates mature NK cell killing of target cells and their production of cytokines and chemokines. This review summarizes the role of NK cells in allograft rejection and proposes mechanistic concepts that indicate a prominent role for KIR-HLA interactions in facilitating NK cells for Fc receptor-mediated ADCC effector function involved in antibody-mediated rejection of solid organ transplants.

  18. Clinical features, course and prognosis of idiopathic membranous nephropathy depending on the presence of antibodies against M-type phospholipase A2 receptor.

    Science.gov (United States)

    Jatem Escalante, Elías; Segarra Medrano, Alfons; Carnicer Cáceres, Clara; Martín-Gómez, M Adoración; Salcedo Allende, María Teresa; Ostos Roldan, Helena; Agraz Pamplona, Irene

    2015-01-01

    In membranous nephropathy, the presence of antibodies against M-type phospholipase A2 receptor is considered highly specific for idiopathic forms. However, no specific association to a particular clinical profile has been found for such antibodies. To assess potential differences in initial clinical profile, course and prognosis of idiopathic membranous nephropathy depending on the presence of anti-PLA2R antibodies. Eighty-five patients with idiopathic membranous nephropathy were included (55 anti-PLA2R-positive and 30 anti-PLA2R-negative). Clinical, biochemical and pathological variables were recorded at the time of diagnosis. Frequency of spontaneous remission, incidence of response to first-line therapy, frequency and number of recurrences, survival of renal function free from renal replacement therapy, survival of renal function free from chronic renal insufficiency and frequency of occurrence of malignant, infectious or autoimmune diseases during follow-up were recorded. At the time of diagnosis, anti-PLA2R-negative patients were significantly older and had a higher frequency of spontaneous remission. No differences were noted in the response to first-line treatment, frequency and number of recurrences, survival of renal function free from renal replacement therapy, or survival of renal function free from chronic renal insufficiency. Anti-PLA2R-negative patients with idiopathic membranous nephropathy were older and experienced spontaneous remission more often than anti-PLA2R-positive patients. No differences in terms of treatment response, recurrences, and final prognosis were observed between both groups of patients. Copyright © 2015 The Authors. Published by Elsevier España, S.L.U. All rights reserved.

  19. Age dependence of spleen- and muscle-corrected hepatic signal enhancement on hepatobiliary phase gadoxetate MRI

    Energy Technology Data Exchange (ETDEWEB)

    Matoori, Simon [Paracelsus Medical University Salzburg, Department of Radiology, Salzburg (Austria); Hirslanden Clinic St. Anna, Clinical Research Group, Lucerne (Switzerland); Froehlich, Johannes M. [Hirslanden Clinic St. Anna, Clinical Research Group, Lucerne (Switzerland); ETH Zurich, Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, Zurich (Switzerland); Cantonal Hospital Winterthur, Department of Radiology, Winterthur (Switzerland); Breitenstein, Stefan [Cantonal Hospital Winterthur, Department of Surgery, Clinic for Visceral and Thoracic Surgery, Winterthur (Switzerland); Doert, Aleksis [Cantonal Hospital Winterthur, Department of Radiology, Winterthur (Switzerland); Pozdniakova, Viktoria [Stavanger University Hospital, Department of Radiology, Stavanger (Norway); Koh, Dow-Mu [Royal Marsden Hospital, Department of Radiology, Surrey, England (United Kingdom); Gutzeit, Andreas [Paracelsus Medical University Salzburg, Department of Radiology, Salzburg (Austria); Hirslanden Clinic St. Anna, Clinical Research Group, Lucerne (Switzerland); Cantonal Hospital Winterthur, Department of Radiology, Winterthur (Switzerland)

    2016-06-15

    To identify correlations of signal enhancements (SE) and SE normalized to reference tissues of the spleen, kidney, liver, musculus erector spinae (MES) and ductus hepatocholedochus (DHC) on hepatobiliary phase gadoxetate-enhanced MRI with patient age in non-cirrhotic patients. A heterogeneous cohort of 131 patients with different clinical backgrounds underwent a standardized 3.0-T gadoxetate-enhanced liver MRI between November 2008 and June 2013. After exclusion of cirrhotic patients, a cohort of 75 patients with no diagnosed diffuse liver disease was selected. The ratio of signal intensity 20 min post- to pre-contrast administration (SE) in the spleen, kidney, liver, MES and DHC, and the SE of the kidney, liver and DHC normalized to the reference tissues spleen or MES were compared to patient age. Patient age was inversely correlated with the liver SE normalized to the spleen and MES SE (both p < 0.001) and proportionally with the SE of the spleen (p = 0.043), the MES (p = 0.030) and the kidney (p = 0.022). No significant correlations were observed for the DHC (p = 0.347) and liver SE (p = 0.606). The age dependence of hepatic SE normalized to the enhancement in the spleen and MES calls for a cautious interpretation of these quantification methods. (orig.)

  20. Enhanced behavioral responses to cold stimuli following CGRPα sensory neuron ablation are dependent on TRPM8.

    Science.gov (United States)

    McCoy, Eric S; Zylka, Mark J

    2014-11-19

    Calcitonin gene-related peptide-α (CGRPα) is a classic marker of peptidergic nociceptive neurons and is expressed in myelinated and unmyelinated dorsal root ganglia (DRG) neurons. Recently, we found that ablation of Cgrpα-expressing sensory neurons reduced noxious heat sensitivity and enhanced sensitivity to cold stimuli in mice. These studies suggested that the enhanced cold responses were due to disinhibition of spinal neurons that receive inputs from cold-sensing/TRPM8 primary afferents; although a direct role for TRPM8 was not examined at the time. Here, we ablated Cgrpα-expressing sensory neurons in mice lacking functional TRPM8 and evaluated sensory responses to noxious heat, cold temperatures, and cold mimetics (acetone evaporative cooling and icilin). We also evaluated thermoregulation in these mice following an evaporative cold challenge. We found that ablation of Cgrpα-expressing sensory neurons in a Trpm8-/- background reduced sensitivity to noxious heat but did not enhance sensitivity to cold stimuli. Thermoregulation following the evaporative cold challenge was not affected by deletion of Trpm8 in control or Cgrpα-expressing sensory neuron-ablated mice. Our data indicate that the enhanced behavioral responses to cold stimuli in CGRPα sensory neuron-ablated mice are dependent on functional TRPM8, whereas the other sensory and thermoregulatory phenotypes caused by CGRPα sensory neuron ablation are independent of TRPM8.

  1. Distance-dependent metal enhanced fluorescence by flowerlike silver nanostructures fabricated in liquid crystalline phase

    Science.gov (United States)

    Zhang, Ying; Yang, Chengliang; Zhang, Guiyang; Peng, Zenghui; Yao, Lishuang; Wang, Qidong; Cao, Zhaoliang; Mu, Quanquan; Xuan, Li

    2017-10-01

    Flowerlike silver nanostructure substrates were fabricated in liquid crystalline phase and the distance dependent property of metal enhanced fluorescence for such substrate was studied for the first time. The distance between silver nanostructures and fluorophore was controlled by the well-established layer-by-layer (LbL) technique constructing alternate layers of poly (allylamine hydrochloride) (PAH) and poly (sodium 4-styrenesulfonate) (PSS). The Rhodamine 6G (R6G) molecules were electrostatically attached to the outmost negative charged PSS layer. The fluorescence enhancement factor of flowerlike nanostructure substrate increased firstly and then decreased with the distance increasing. The best enhanced fluorescence intensity of 71 fold was obtained at a distance of 5.2 nm from the surface of flowerlike silver nanostructure. The distance for best enhancement effect is an instructive parameter for the applications of such substrates and could be used in the practical MEF applications with the flowerlike nanostructure substrates fabricated in such way which is simple, controllable and cost-effective.

  2. Influenza Vaccination in the Face of Maternal Antibody Results in Enhanced Pneumonia Following Heterologous, Homosubtypic Influenza Challenge

    Science.gov (United States)

    Inactivated influenza vaccines do not provide cross-protection to diverse antigenic strains that could be circulating or suddenly arise in a population. It is desirable to vaccinate at a young age to provide maximum protection from influenza; however, maternally-derived factors (antibody or cells) c...

  3. Site-specific conjugation of single domain antibodies to liposomes enhances photosensitizer uptake and photodynamic therapy efficacy

    NARCIS (Netherlands)

    Broekgaarden, M.; van Vught, R.; Oliveira, S.; Roovers, R. C.; van Bergen En Henegouwen, P. M. P.; Pieters, R. J.; van Gulik, T. M.; Breukink, E.; Heger, M.

    2016-01-01

    Photodynamic therapy for therapy-resistant cancers will greatly benefit from targeted delivery of tumor photosensitizing agents. In this study, a strategy for the site-specific conjugation of single domain antibodies onto liposomes containing the photosensitizer zinc phthalocyanine was developed and

  4. Site-specific conjugation of single domain antibodies to liposomes enhances photosensitizer uptake and photodynamic therapy efficacy.

    Science.gov (United States)

    Broekgaarden, M; van Vught, R; Oliveira, S; Roovers, R C; van Bergen en Henegouwen, P M P; Pieters, R J; Van Gulik, T M; Breukink, E; Heger, M

    2016-03-28

    Photodynamic therapy for therapy-resistant cancers will greatly benefit from targeted delivery of tumor photosensitizing agents. In this study, a strategy for the site-specific conjugation of single domain antibodies onto liposomes containing the photosensitizer zinc phthalocyanine was developed and tested.

  5. Bacterial endotoxin enhances colorectal cancer cell adhesion and invasion through TLR-4 and NF-kappaB-dependent activation of the urokinase plasminogen activator system.

    LENUS (Irish Health Repository)

    Killeen, S D

    2009-05-19

    Perioperative exposure to lipopolysaccharide (LPS) is associated with accelerated metastatic colorectal tumour growth. LPS directly affects cells through Toll-like receptor 4 (TLR-4) and the transcription factor NF-kappaB. The urokinase plasminogen activator (u-PA) system is intimately implicated in tumour cell extracellular matrix (ECM) interactions fundamental to tumour progression. Thus we sought to determine if LPS directly induces accelerated tumour cell ECM adhesion and invasion through activation of the u-PA system and to elucidate the cellular pathways involved. Human colorectal tumour cell lines were stimulated with LPS. u-PA concentration, u-PA activity, active u-PA, surface urokinase plasminogen activator receptor (u-PAR) and TLR-4 expression were assessed by ELISA, colorimetric assay, western blot analysis and flow cytometry respectively. In vitro tumour cell vitronectin adhesion and ECM invasion were analysed by vitronectin adhesion assay and ECM invasion chambers. u-PA and u-PAR function was inhibited with anti u-PA antibodies or the selective u-PA inhibitors amiloride or WXC-340, TLR-4 by TLR-4-blocking antibodies and NF-kappaB by the selective NF-kappaB inhibitor SN-50. LPS upregulates u-PA and u-PAR in a dose-dependent manner, enhancing in vitro tumour cell vitronectin adhesion and ECM invasion by >40% (P<0.01). These effects were ameliorated by u-PA and u-PAR inhibition. LPS activates NF-kappaB through TLR-4. TLR-4 and NF-kappaB inhibition ameliorated LPS-enhanced u-PA and u-PAR expression, tumour cell vitronectin adhesion and ECM invasion. LPS promotes tumour cell ECM adhesion and invasion through activation of the u-PA system in a TLR-4- and NF-kappaB-dependent manner.

  6. A photosensitizer delivered by bispecific antibody redirected T lymphocytes enhances cytotoxicity against EpCAM-expressing carcinoma cells upon light irradiation.

    Science.gov (United States)

    Blaudszun, André-René; Moldenhauer, Gerhard; Schneider, Marc; Philippi, Anja

    2015-01-10

    Recently conducted clinical trials have provided impressive evidence that chemotherapy resistant metastatic melanoma and several hematological malignancies can be cured using adoptive T cell therapy or T cell-recruiting bispecific antibodies. However, a significant fraction of patients did not benefit from these treatments. Here we have evaluated the feasibility of a novel combination therapy which aims to further enhance the killing potential of bispecific antibody-redirected T lymphocytes by using these cells as targeted delivery system for photosensitizing agents. For a first in vitro proof-of-concept study, ex vivo activated human donor T cells were loaded with a poly(styrene sulfonate) (PSS)-complex of the model photosensitizer 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin (mTHPP). In the absence of light and when loading with the water-soluble PSS/mTHPP-complex occurred at a tolerable concentration, viability and cytotoxic function of loaded T lymphocytes were not impaired. When "drug-enhanced" T cells were co-cultivated with EpCAM-expressing human carcinoma cells, mTHPP was transferred to target cells. Notably, in the presence of a bispecific antibody, which cross-links effector and target cells thereby inducing the cytolytic activity of cytotoxic T lymphocytes, significantly more photosensitizer was transferred. Consequently, upon irradiation of co-cultures, redirected drug-loaded T cells were more effective in killing A549 lung and SKOV-3 ovarian carcinoma cells than retargeted unloaded T lymphocytes. Particularly, the additive approach using redirected unloaded T cells in combination with appropriate amounts of separately applied PSS/mTHPP was less efficient as well. Thus, by loading T lymphocytes with a stimulus-sensitive anti-cancer drug, we were able to enhance the cytotoxic capacity of carrier cells. Photosensitizer boosted T cells could open new perspectives for adoptive T cell therapy as well as targeted photodynamic therapy. Copyright © 2014

  7. Sex-dependent immune responses to infant vaccination: an individual participant data meta-analysis of antibody and memory B cells.

    Science.gov (United States)

    Voysey, Merryn; Barker, Charlotte I S; Snape, Matthew D; Kelly, Dominic F; Trück, Johannes; Pollard, Andrew J

    2016-03-29

    Biological sex can be an important source of variation in infection and immunity and sex-dependent differences in immune response to vaccination have been reported in some studies. We conducted an individual participant data meta-analysis of vaccine trials from one research centre, in which vaccines were administered to children under three years of age and immunological parameters measured. Log-transformed antigen-specific antibody and memory B cell results were meta-analysed and differences between girls and boys reported as geometric mean ratios. Antibody and memory B cell data were available from nine trials and 2378 children. Statistically significant differences between girls and boys were observed for diphtheria toxoid, capsular group A, W, and Y meningococcal, and pneumococcal vaccines. No sex-differences were observed for responses to Haemophilus influenzae type b, capsular group C meningococcal or tetanus toxoid vaccines. In young children, immune responses to vaccines were consistently higher or equivalent in girls compared with boys. In no instance were responses in boys significantly higher than girls. While these data do not indicate differences in protection conferred by immunisation in boys and girls, they do support further consideration of biological sex in planning of clinical trials of vaccines. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Characterizing the Effects of Intermittent Faults on a Processor for Dependability Enhancement Strategy

    Directory of Open Access Journals (Sweden)

    Chao(Saul Wang

    2014-01-01

    Full Text Available As semiconductor technology scales into the nanometer regime, intermittent faults have become an increasing threat. This paper focuses on the effects of intermittent faults on NET versus REG on one hand and the implications for dependability strategy on the other. First, the vulnerability characteristics of representative units in OpenSPARC T2 are revealed, and in particular, the highly sensitive modules are identified. Second, an arch-level dependability enhancement strategy is proposed, showing that events such as core/strand running status and core-memory interface events can be candidates of detectable symptoms. A simple watchdog can be deployed to detect application running status (IEXE event. Then SDC (silent data corruption rate is evaluated demonstrating its potential. Third and last, the effects of traditional protection schemes in the target CMT to intermittent faults are quantitatively studied on behalf of the contribution of each trap type, demonstrating the necessity of taking this factor into account for the strategy.

  9. Characterizing the effects of intermittent faults on a processor for dependability enhancement strategy.

    Science.gov (United States)

    Wang, Chao Saul; Fu, Zhong-Chuan; Chen, Hong-Song; Wang, Dong-Sheng

    2014-01-01

    As semiconductor technology scales into the nanometer regime, intermittent faults have become an increasing threat. This paper focuses on the effects of intermittent faults on NET versus REG on one hand and the implications for dependability strategy on the other. First, the vulnerability characteristics of representative units in OpenSPARC T2 are revealed, and in particular, the highly sensitive modules are identified. Second, an arch-level dependability enhancement strategy is proposed, showing that events such as core/strand running status and core-memory interface events can be candidates of detectable symptoms. A simple watchdog can be deployed to detect application running status (IEXE event). Then SDC (silent data corruption) rate is evaluated demonstrating its potential. Third and last, the effects of traditional protection schemes in the target CMT to intermittent faults are quantitatively studied on behalf of the contribution of each trap type, demonstrating the necessity of taking this factor into account for the strategy.

  10. The Human Antibody Response to Dengue Virus Infection

    Directory of Open Access Journals (Sweden)

    Aravinda M. de Silva

    2011-11-01

    Full Text Available Dengue viruses (DENV are the causative agents of dengue fever (DF and dengue hemorrhagic fever (DHF. Here we review the current state of knowledge about the human antibody response to dengue and identify important knowledge gaps. A large body of work has demonstrated that antibodies can neutralize or enhance DENV infection. Investigators have mainly used mouse monoclonal antibodies (MAbs to study interactions between DENV and antibodies. These studies indicate that antibody neutralization of DENVs is a “multi-hit” phenomenon that requires the binding of multiple antibodies to neutralize a virion. The most potently neutralizing mouse MAbs bind to surface exposed epitopes on domain III of the dengue envelope (E protein. One challenge facing the dengue field now is to extend these studies with mouse MAbs to better understand the human antibody response. The human antibody response is complex as it involves a polyclonal response to primary and secondary infections with 4 different DENV serotypes. Here we review studies conducted with immune sera and MAbs isolated from people exposed to dengue infections. Most dengue-specific antibodies in human immune sera are weakly neutralizing and bind to multiple DENV serotypes. The human antibodies that potently and type specifically neutralize DENV represent a small fraction of the total DENV-specific antibody response. Moreover, these neutralizing antibodies appear to bind to novel epitopes including complex, quaternary epitopes that are only preserved on the intact virion. These studies establish that human and mouse antibodies recognize distinct epitopes on the dengue virion. The leading theory proposed to explain the increased risk of severe disease in secondary cases is antibody dependent enhancement (ADE, which postulates that weakly neutralizing antibodies from the first infection bind to the second serotype and enhance infection of FcγR bearing myeloid cells such as monocytes and macrophages. Here

  11. The HIV1 protein Vpr acts to enhance constitutive DCAF1-dependent UNG2 turnover.

    Science.gov (United States)

    Wen, Xiaoyun; Casey Klockow, Laurieann; Nekorchuk, Michael; Sharifi, Hamayun J; de Noronha, Carlos M C

    2012-01-01

    The HIV1 protein Vpr assembles with and acts through an ubiquitin ligase complex that includes DDB1 and cullin 4 (CRL4) to cause G2 cell cycle arrest and to promote degradation of both uracil DNA glycosylase 2 (UNG2) and single-strand selective mono-functional uracil DNA glycosylase 1 (SMUG1). DCAF1, an adaptor protein, is required for Vpr-mediated G2 arrest through the ubiquitin ligase complex. In work described here, we used UNG2 as a model substrate to study how Vpr acts through the ubiquitin ligase complex. We examined whether DCAF1 is essential for Vpr-mediated degradation of UNG2 and SMUG1. We further investigated whether Vpr is required for recruiting substrates to the ubiquitin ligase or acts to enhance its function and whether this parallels Vpr-mediated G2 arrest. We found that DCAF1 plays an important role in Vpr-independent UNG2 and SMUG1 depletion. UNG2 assembled with the ubiquitin ligase complex in the absence of Vpr, but Vpr enhanced this interaction. Further, Vpr-mediated enhancement of UNG2 degradation correlated with low Vpr expression levels. Vpr concentrations exceeding a threshold blocked UNG2 depletion and enhanced its accumulation in the cell nucleus. A similar dose-dependent trend was seen for Vpr-mediated cell cycle arrest. This work identifies UNG2 and SMUG1 as novel targets for CRL4(DCAF1)-mediated degradation. It further shows that Vpr enhances rather than enables the interaction between UNG2 and the ubiquitin ligase. Vpr augments CRL4(DCAF1)-mediated UNG2 degradation at low concentrations but antagonizes it at high concentrations, allowing nuclear accumulation of UNG2. Further, the protein that is targeted to cause G2 arrest behaves much like UNG2. Our findings provide the basis for determining whether the CRL4(DCAF1) complex is alone responsible for cell cycle-dependent UNG2 turnover and will also aid in establishing conditions necessary for the identification of additional targets of Vpr-enhanced degradation.

  12. The HIV1 protein Vpr acts to enhance constitutive DCAF1-dependent UNG2 turnover.

    Directory of Open Access Journals (Sweden)

    Xiaoyun Wen

    Full Text Available The HIV1 protein Vpr assembles with and acts through an ubiquitin ligase complex that includes DDB1 and cullin 4 (CRL4 to cause G2 cell cycle arrest and to promote degradation of both uracil DNA glycosylase 2 (UNG2 and single-strand selective mono-functional uracil DNA glycosylase 1 (SMUG1. DCAF1, an adaptor protein, is required for Vpr-mediated G2 arrest through the ubiquitin ligase complex. In work described here, we used UNG2 as a model substrate to study how Vpr acts through the ubiquitin ligase complex. We examined whether DCAF1 is essential for Vpr-mediated degradation of UNG2 and SMUG1. We further investigated whether Vpr is required for recruiting substrates to the ubiquitin ligase or acts to enhance its function and whether this parallels Vpr-mediated G2 arrest.We found that DCAF1 plays an important role in Vpr-independent UNG2 and SMUG1 depletion. UNG2 assembled with the ubiquitin ligase complex in the absence of Vpr, but Vpr enhanced this interaction. Further, Vpr-mediated enhancement of UNG2 degradation correlated with low Vpr expression levels. Vpr concentrations exceeding a threshold blocked UNG2 depletion and enhanced its accumulation in the cell nucleus. A similar dose-dependent trend was seen for Vpr-mediated cell cycle arrest.This work identifies UNG2 and SMUG1 as novel targets for CRL4(DCAF1-mediated degradation. It further shows that Vpr enhances rather than enables the interaction between UNG2 and the ubiquitin ligase. Vpr augments CRL4(DCAF1-mediated UNG2 degradation at low concentrations but antagonizes it at high concentrations, allowing nuclear accumulation of UNG2. Further, the protein that is targeted to cause G2 arrest behaves much like UNG2. Our findings provide the basis for determining whether the CRL4(DCAF1 complex is alone responsible for cell cycle-dependent UNG2 turnover and will also aid in establishing conditions necessary for the identification of additional targets of Vpr-enhanced degradation.

  13. Radiolabeling and biotinylation of internalizing monoclonal antibody chimeric BR96: Potential use of extracorporeal immunoadsorption with enhanced tumor radioactivity retention of Iodine, Indium and Rhenium

    Energy Technology Data Exchange (ETDEWEB)

    Chen, JianQing

    1997-01-01

    In this thesis, methodology of radiolabeling and simultaneous biotinylation for internalizing monoclonal antibody chimeric BR96 have been investigated by using three element groups of potential therapeutic radionuclides iodine, indium and rhenium, and their different labeling methods. The biodistribution and kinetics of biotinylated and radiolabeled chiBR96 have been studied in colon carcinoma isografted rats. The potential use of ECIA, based on the biotin-avidin concept, has been evaluated and compared with the approach of avidin `chase` in the same animal tumor model with respect to an enhancement of tumor-to-normal tissue (T/N) activity ratio. 131 refs.

  14. Radiolabeling and biotinylation of internalizing monoclonal antibody chimeric BR96: Potential use of extracorporeal immunoadsorption with enhanced tumor radioactivity retention of Iodine, Indium and Rhenium

    International Nuclear Information System (INIS)

    Chen, JianQing.

    1997-01-01

    In this thesis, methodology of radiolabeling and simultaneous biotinylation for internalizing monoclonal antibody chimeric BR96 have been investigated by using three element groups of potential therapeutic radionuclides iodine, indium and rhenium, and their different labeling methods. The biodistribution and kinetics of biotinylated and radiolabeled chiBR96 have been studied in colon carcinoma isografted rats. The potential use of ECIA, based on the biotin-avidin concept, has been evaluated and compared with the approach of avidin 'chase' in the same animal tumor model with respect to an enhancement of tumor-to-normal tissue (T/N) activity ratio. 131 refs

  15. Age-dependent differences in cytokine and antibody responses after experimental RSV infection in a bovine model

    DEFF Research Database (Denmark)

    Grell, S.N.; Riber, Ulla; Tjørnehøj, Kirsten

    2005-01-01

    Respiratory syncytial virus (RSV) causes severe respiratory disease in both infants and calves. As in humans, bovine RSV (BRSV) infections are most severe in the first 6 months of life. In this study, experimental infection with BRSV was performed in calves aged 1-5, 9-16 or 32-37 weeks. Compared...... "inflammatory" but weak humoral antiviral response in very young animals suggests that enhanced inflammation contributes to disease during RSV infection during the early postnatal period....

  16. Effect of anti-carbohydrate antibodies on HIV infection in a monocytic cell line (U937)

    DEFF Research Database (Denmark)

    Hansen, J E; Nielsen, C; Clausen, H

    1991-01-01

    Monoclonal antibodies (mAbs) against carbohydrate epitopes of gp120 have recently been found to inhibit HIV infection of lymphocytes in vitro thereby opening new possibilities for vaccine considerations. Antibody-dependent enhancement of infection has however come increasingly into focus....... This study therefore investigated the neutralization of HIV in a monocytic cell line (U937) using mAbs against these carbohydrate gp120-epitopes. While antibodies against one of the epitopes (AI) neutralized infection of U937 cells despite binding to the Fc-receptor, one mAb against the sialosyl-Tn epitope...... enhanced infection. This enhancement was independent of complement and could be blocked by mAb Leu3a against the CD4-receptor. The study indicated that enhancement of infection in monocytic cells can occur by the same anti-carbohydrate antibodies that neutralize infection in lymphocytes, and that antibody...

  17. Type I CD20 Antibodies Recruit the B Cell Receptor for Complement-Dependent Lysis of Malignant B Cells

    NARCIS (Netherlands)

    Engelberts, Patrick J.; Voorhorst, Marleen; Schuurman, Janine; van Meerten, Tom; Bakker, Joost M.; Vink, Tom; Mackus, Wendy J. M.; Breij, Esther C. W.; Derer, Stefanie; Valerius, Thomas; van de Winkel, Jan G. J.; Parren, Paul W. H. I.; Beurskens, Frank J.

    2016-01-01

    Human IgG1 type I CD20 Abs, such as rituximab and ofatumumab (OFA), efficiently induce complement-dependent cytotoxicity (CDC) of CD20(+) B cells by binding of C1 to hexamerized Fc domains. Unexpectedly, we found that type I CD20 Ab F(ab ')2 fragments, as well as C1q-binding-deficient IgG mutants,

  18. HIV-1 envelope glycoprotein resistance to monoclonal antibody 2G12 is subject-specific and context-dependent in macaques and humans.

    Directory of Open Access Journals (Sweden)

    Delphine C Malherbe

    Full Text Available HIV-1 Envelope (Env protein is the sole target of neutralizing antibodies (NAbs that arise during infection to neutralize autologous variants. Under this immune pressure, HIV escape variants are continuously selected and over the course of infection Env becomes more neutralization resistant. Many common alterations are known to affect sensitivity to NAbs, including residues encoding potential N-linked glycosylation sites (PNGS. Knowledge of Env motifs associated with neutralization resistance is valuable for the design of an effective Env-based vaccine so we characterized Envs isolated longitudinally from a SHIV(SF162P4 infected macaque for sensitivity to neutralizing monoclonal antibodies (MAbs B12, 2G12, 4E10 and 2F5. The early Env, isolated from plasma at day 56 after infection, was the most sensitive and the late Env, from day 670, was the most resistant to MAbs. We identified four PNGS in these Envs that accumulated over time at positions 130, 139, 160 and 397. We determined that removal of these PNGS significantly increased neutralization sensitivity to 2G12, and conversely, we identified mutations by in silico analyses that contributed resistance to 2G12 neutralization. In order to expand our understanding of these PNGS, we analyzed Envs from clade B HIV-infected human subjects and identified additional glycan and amino acid changes that could affect neutralization by 2G12 in a context-dependent manner. Taken together, these in vitro and in silico analyses of clade B Envs revealed that 2G12 resistance is achieved by previously unrecognized PNGS substitutions in a context-dependent manner and by subject-specific pathways.

  19. Refractive index dependent local electric field enhancement in cylindrical gold nanohole

    International Nuclear Information System (INIS)

    Zhu Jian

    2011-01-01

    We report on the local electric field characters in a long cylindrical gold nanohole. Theoretical calculation results based on quasi-static model show that the local environmental dielectric constant dependent electric field intensity and field distribution in the gold nanohole show quite unique properties, different from those in the thin gold nanotube. Because of the thick gold wall, no plasmon hybridization exists. So there is only one resonance frequency taking place, and the intense local field has been focused into the gold nanohole. Our main finding is that, the local field in the nanohole is largely dependent on the inner hole refractive index and outer environmental refractive index. The competition between inner hole and outer polarization leads to a non-monotonic change of the local field intensity with increasing the dielectric constant of the nanohole. This refractive index controlled local field enhancement in cylindrical gold nanohole presents a potential for tunable surface-enhanced fluorescence and novel nano-optical biosensing applications.

  20. Bioorthogonal, two-component delivery systems based on antibody and drug-loaded nanocarriers for enhanced internalization of nanotherapeutics

    OpenAIRE

    Hapuarachchige, Sudath; Zhu, Wenlian; Kato, Yoshinori; Artemov, Dmitri

    2013-01-01

    Nanocarriers play an important role in targeted cancer chemotherapy. The optimal nanocarrier delivery system should provide efficient and highly specific recognition of the target cells and rapid internalization of the therapeutic cargo to reduce systemic toxicity as well as to increase the cytotoxicity to cancer cells. To this end, we developed a two-step, two-component targeted delivery system based on antibody and drug-loaded nanocarrier that uses bioorthogonal click reactions for specific...

  1. Predicting stimulation-dependent enhancer-promoter interactions from ChIP-Seq time course data.

    Science.gov (United States)

    Dzida, Tomasz; Iqbal, Mudassar; Charapitsa, Iryna; Reid, George; Stunnenberg, Henk; Matarese, Filomena; Grote, Korbinian; Honkela, Antti; Rattray, Magnus

    2017-01-01

    We have developed a machine learning approach to predict stimulation-dependent enhancer-promoter interactions using evidence from changes in genomic protein occupancy over time. The occupancy of estrogen receptor alpha (ERα), RNA polymerase (Pol II) and histone marks H2AZ and H3K4me3 were measured over time using ChIP-Seq experiments in MCF7 cells stimulated with estrogen. A Bayesian classifier was developed which uses the correlation of temporal binding patterns at enhancers and promoters and genomic proximity as features to predict interactions. This method was trained using experimentally determined interactions from the same system and was shown to achieve much higher precision than predictions based on the genomic proximity of nearest ERα binding. We use the method to identify a genome-wide confident set of ERα target genes and their regulatory enhancers genome-wide. Validation with publicly available GRO-Seq data demonstrates that our predicted targets are much more likely to show early nascent transcription than predictions based on genomic ERα binding proximity alone.

  2. Predicting stimulation-dependent enhancer-promoter interactions from ChIP-Seq time course data

    Directory of Open Access Journals (Sweden)

    Tomasz Dzida

    2017-09-01

    Full Text Available We have developed a machine learning approach to predict stimulation-dependent enhancer-promoter interactions using evidence from changes in genomic protein occupancy over time. The occupancy of estrogen receptor alpha (ERα, RNA polymerase (Pol II and histone marks H2AZ and H3K4me3 were measured over time using ChIP-Seq experiments in MCF7 cells stimulated with estrogen. A Bayesian classifier was developed which uses the correlation of temporal binding patterns at enhancers and promoters and genomic proximity as features to predict interactions. This method was trained using experimentally determined interactions from the same system and was shown to achieve much higher precision than predictions based on the genomic proximity of nearest ERα binding. We use the method to identify a genome-wide confident set of ERα target genes and their regulatory enhancers genome-wide. Validation with publicly available GRO-Seq data demonstrates that our predicted targets are much more likely to show early nascent transcription than predictions based on genomic ERα binding proximity alone.

  3. Gadodiamide injection for enhancement of MRI in the CNS. Applications, dose, field and time dependence

    International Nuclear Information System (INIS)

    Aakeson, P.

    1996-01-01

    Gadodiamide injection was comparable to Gd-DTPA with regard to both safety and diagnostic efficiency in the central nervous system. The contrast effect of Gd contrast agents is higher at 1.5 T than at 0.3 T both in phantoms and patients with a maximum ratio (signal lesion/signal grey matter) more than 50% higher at 1.5 T. To achieve high contrast effect, heavily T1-weighted images are important. Prolonging the TR from 400 ms to 600 ms reduced the ratio by 15-45% depending on concentration. The effective time window for imaging of BBB (Blood-Brain Barrier) damage is between 2-5 and 25-30 minutes after injection and several scans can be performed without loss of enhancement. To provide maximum detectability of BBB damage in patients, higher doses of Gd contrast media should be useful, especially at low field strengths, as the doses used clinically today do not utilize the maximum contrast effect. High-dose (0.3 mmol/kg b.w.) contrast enhanced MRI (0.3 T) with Gadodiamide injection allowed detection of significantly more and smaller metastases (i.e. BBB damage) than standard dose (0.1 mmol/kg b.w.) High dose contrast-enhanced MRI (0.3 T) did not increase the diagnostic information for the evaluation of patients with failed back surgery syndrome compared to standard dose MRI. 55 refs, 9 figs, 10 tabs

  4. Gadodiamide injection for enhancement of MRI in the CNS. Applications, dose, field and time dependence

    Energy Technology Data Exchange (ETDEWEB)

    Aakeson, P.

    1996-10-01

    Gadodiamide injection was comparable to Gd-DTPA with regard to both safety and diagnostic efficiency in the central nervous system. The contrast effect of Gd contrast agents is higher at 1.5 T than at 0.3 T both in phantoms and patients with a maximum ratio (signal lesion/signal grey matter) more than 50% higher at 1.5 T. To achieve high contrast effect, heavily T1-weighted images are important. Prolonging the TR from 400 ms to 600 ms reduced the ratio by 15-45% depending on concentration. The effective time window for imaging of BBB (Blood-Brain Barrier) damage is between 2-5 and 25-30 minutes after injection and several scans can be performed without loss of enhancement. To provide maximum detectability of BBB damage in patients, higher doses of Gd contrast media should be useful, especially at low field strengths, as the doses used clinically today do not utilize the maximum contrast effect. High-dose (0.3 mmol/kg b.w.) contrast enhanced MRI (0.3 T) with Gadodiamide injection allowed detection of significantly more and smaller metastases (i.e. BBB damage) than standard dose (0.1 mmol/kg b.w.) High dose contrast-enhanced MRI (0.3 T) did not increase the diagnostic information for the evaluation of patients with failed back surgery syndrome compared to standard dose MRI. 55 refs, 9 figs, 10 tabs.

  5. Morphology-dependent enhancement of the pseudocapacitance of template-guided tunable polyaniline nanostructures

    KAUST Repository

    Chen, Wei

    2013-07-25

    Polyaniline is one of the most investigated conducting polymers as supercapacitor material for energy storage applications. The preparation of nanostructured polyaniline with well-controlled morphology is crucial to obtaining good supercapacitor performance. We present here a facile chemical process to produce polyaniline nanostructures with three different morphologies (i.e., nanofibers, nanospheres, and nanotubes) by utilizing the corresponding tunable morphology of MnO2 reactive templates. A growth mechanism is proposed to explain the evolution of polyaniline morphology based on the reactive templates. The morphology-induced improvement in the electrochemical performance of polyaniline pseudocapacitors is as large as 51% due to the much enhanced surface area and the porous nature of the template-guided polyaniline nanostructures. In addition, and for the first time, a redox-active electrolyte is applied to the polyaniline pseudocapacitors to achieve significant enhancement of pseudocapacitance. Compared to the conventional electrolyte, the enhancement of pseudocapacitance in the redox-active electrolyte is 49%-78%, depending on the specific polyaniline morphology, reaching the highest reported capacitance of 896 F/g for polyaniline full cells so far. © 2013 American Chemical Society.

  6. Nitric oxide donors enhance the frequency dependence of dopamine release in nucleus accumbens.

    Science.gov (United States)

    Hartung, Henrike; Threlfell, Sarah; Cragg, Stephanie J

    2011-08-01

    Dopamine (DA) neurotransmission in the nucleus accumbens (NAc) is critically involved in normal as well as maladaptive motivated behaviors including drug addiction. Whether the striatal neuromodulator nitric oxide (NO) influences DA release in NAc is unknown. We investigated whether exogenous NO modulates DA transmission in NAc core and how this interaction varies depending on the frequency of presynaptic activation. We detected DA with cyclic voltammetry at carbon-fiber microelectrodes in mouse NAc in slices following stimuli spanning a full range of DA neuron firing frequencies (1-100 Hz). NO donors 3-morpholinosydnonimine hydrochloride (SIN-1) or z-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate (PAPA/NONOate) enhanced DA release with increasing stimulus frequency. This NO-mediated enhancement of frequency sensitivity of DA release was not prevented by inhibition of soluble guanylyl cyclase (sGC), DA transporters, or large conductance Ca(2+)-activated K(+) channels, and did not require glutamatergic or GABAergic input. However, experiments to identify whether frequency-dependent NO effects were mediated via changes in powerful acetylcholine-DA interactions revealed multiple components to NO modulation of DA release. In the presence of a nicotinic receptor antagonist (dihydro-β-erythroidine), NO donors increased DA release in a frequency-independent manner. These data suggest that NO in the NAc can modulate DA release through multiple GC-independent neuronal mechanisms whose net outcome varies depending on the activity in DA neurons and accumbal cholinergic interneurons. In the presence of accumbal acetylcholine, NO promotes the sensitivity of DA release to presynaptic activation, but with reduced acetylcholine input, NO will promote DA release in an activity-independent manner through a direct action on dopaminergic terminals.

  7. Spinal cord injury-induced immune deficiency syndrome enhances infection susceptibility dependent on lesion level.

    Science.gov (United States)

    Brommer, Benedikt; Engel, Odilo; Kopp, Marcel A; Watzlawick, Ralf; Müller, Susanne; Prüss, Harald; Chen, Yuying; DeVivo, Michael J; Finkenstaedt, Felix W; Dirnagl, Ulrich; Liebscher, Thomas; Meisel, Andreas; Schwab, Jan M

    2016-03-01

    Pneumonia is the leading cause of death after acute spinal cord injury and is associated with poor neurological outcome. In contrast to the current understanding, attributing enhanced infection susceptibility solely to the patient's environment and motor dysfunction, we investigate whether a secondary functional neurogenic immune deficiency (spinal cord injury-induced immune deficiency syndrome, SCI-IDS) may account for the enhanced infection susceptibility. We applied a clinically relevant model of experimental induced pneumonia to investigate whether the systemic SCI-IDS is functional sufficient to cause pneumonia dependent on spinal cord injury lesion level and investigated whether findings are mirrored in a large prospective cohort study after human spinal cord injury. In a mouse model of inducible pneumonia, high thoracic lesions that interrupt sympathetic innervation to major immune organs, but not low thoracic lesions, significantly increased bacterial load in lungs. The ability to clear the bacterial load from the lung remained preserved in sham animals. Propagated immune susceptibility depended on injury of central pre-ganglionic but not peripheral postganglionic sympathetic innervation to the spleen. Thoracic spinal cord injury level was confirmed as an independent increased risk factor of pneumonia in patients after motor complete spinal cord injury (odds ratio = 1.35, P spinal cord injury directly causes increased risk for bacterial infection in mice as well as in patients. Besides obvious motor and sensory paralysis, spinal cord injury also induces a functional SCI-IDS ('immune paralysis'), sufficient to propagate clinically relevant infection in an injury level dependent manner. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  8. Enhanced coal-dependent methanogenesis coupled with algal biofuels: Potential water recycle and carbon capture

    Science.gov (United States)

    Barnhart, Elliott P.; Davis, Katherine J.; Varonka, Matthew; Orem, William H.; Cunningham, Alfred B.; Ramsay, Bradley D.; Fields, Matthew W.

    2017-01-01

    Many coal beds contain microbial communities that can convert coal to natural gas (coalbed methane). Native microorganisms were obtained from Powder River Basin (PRB) coal seams with a diffusive microbial sampler placed downhole and used as an inoculum for enrichments with different nutrients to investigate microbially-enhanced coalbed methane production (MECoM). Coal-dependent methanogenesis more than doubled when yeast extract (YE) and several less complex components (proteins and amino acids) were added to the laboratory microcosms. Stimulated coal-dependent methanogenesis with peptone was 86% of that with YE while glutamate-stimulated activity was 65% of that with YE, and a vitamin mix had only 33% of the YE stimulated activity. For field application of MECoM, there is interest in identifying cost-effective alternatives to YE and other expensive nutrients. In laboratory studies, adding algal extract (AE) with lipids removed stimulated coal-dependent methanogenesis and the activity was 60% of that with YE at 27 d and almost 90% of YE activity at 1406 d. Analysis of British Thermal Unit (BTU) content of coal (a measure of potential energy yield) from long-term incubations indicated > 99.5% of BTU content remained after coalbed methane (CBM) stimulation with either AE or YE. Thus, the coal resource remains largely unchanged following stimulated microbial methane production. Algal CBM stimulation could lead to technologies that utilize coupled biological systems (photosynthesis and methane production) that sustainably enhance CBM production and generate algal biofuels while also sequestering carbon dioxide (CO2).

  9. Blocking Tumor Necrosis Factor α Enhances CD8 T-cell-Dependent Immunity in Experimental Melanoma.

    Science.gov (United States)

    Bertrand, Florie; Rochotte, Julia; Colacios, Céline; Montfort, Anne; Tilkin-Mariamé, Anne-Françoise; Touriol, Christian; Rochaix, Philippe; Lajoie-Mazenc, Isabelle; Andrieu-Abadie, Nathalie; Levade, Thierry; Benoist, Hervé; Ségui, Bruno

    2015-07-01

    TNF plays a dual, still enigmatic role in melanoma, either acting as a cytotoxic cytokine or favoring a tumorigenic inflammatory microenvironment. Herein, the tumor growth of melanoma cell lines expressing major histocompatibility complex class I molecules at high levels (MHC-I(high)) was dramatically impaired in TNF-deficient mice, and this was associated with enhanced tumor-infiltrating CD8(+) T lymphocytes. Immunodepletion of CD8 T cells fully restored melanoma growth in TNF(-/-) mice. Systemic administration of Etanercept inhibited MHC-I(high) melanoma growth in immunocompetent but not in immunodeficient (IFNγ(-/-), nude, or CD8(-/-)) mice. MHC-I(high) melanoma growth was also reduced in mice lacking TNF-R1, but not TNF-R2. TNF(-/-) and TNF-R1(-/-) mice as well as Etanercept-treated WT mice displayed enhanced intratumor content of high endothelial venules surrounded by high CD8(+) T-cell density. Adoptive transfer of activated TNF-R1-deficient or -proficient CD8(+) T cells in CD8-deficient mice bearing B16K1 tumors demonstrated that TNF-R1 deficiency facilitates the accumulation of live CD8(+) T cells into the tumors. Moreover, in vitro experiments indicated that TNF triggered activated CD8(+) T cell death in a TNF-R1-dependent manner, likely limiting the accumulation of tumor-infiltrating CD8(+) T cells in TNF/TNF-R1-proficient animals. Collectively, our observations indicate that TNF-R1-dependent TNF signaling impairs tumor-infiltrating CD8(+) T-cell accumulation and may serve as a putative target to favor CD8(+) T-cell-dependent immune response in melanoma. ©2015 American Association for Cancer Research.

  10. Neuron-astrocyte interaction enhance GABAergic synaptic transmission in a manner dependent on key metabolic enzymes.

    Directory of Open Access Journals (Sweden)

    Przemysław eKaczor

    2015-04-01

    Full Text Available GABA is the major inhibitory neurotransmitter in the adult brain and mechanisms of GABAergic inhibition have been intensely investigated in the past decades. Recent studies provided evidence for an important role of astrocytes in shaping GABAergic currents. One of the most obvious, but yet poorly understood, mechanisms of the cross-talk between GABAergic currents and astrocytes is metabolism including neurotransmitter homeostasis. In particular, how modulation of GABAergic currents by astrocytes depends on key enzymes involved in cellular metabolism remains largely unknown. To address this issue, we have considered two simple models of neuronal cultures: nominally astrocyte-free neuronal culture (NC and neuronal-astrocytic co-cultures (ANCC and miniature Inhibitory Postsynaptic Currents (mIPSCs were recorded in control conditions and in the presence of respective enzyme blockers. We report that enrichment of neuronal culture with astrocytes results in a marked increase in mIPSC frequency. This enhancement of GABAergic activity was accompanied by increased number of GAD65 and vGAT puncta, indicating that at least a part of the frequency enhancement was due to increased number of synaptic contacts. Inhibition of glutamine synthetase (with MSO strongly reduced mIPSC frequency in ANCC but had no effect in NC. Moreover, treatment of ANCC with inhibitor of glycogen phosphorylase (BAYU6751 or with selective inhibitor of astrocytic Krebs cycle,fluoroacetate, resulted in a marked reduction of mIPSC frequency in ANCC having no effect in NC. We conclude that GABAergic synaptic transmission strongly depends on neuron-astrocyte interaction in a manner dependent on key metabolic enzymes as well as on the Krebs cycle.

  11. Enhanced radiosensitization of enzalutamide via schedule dependent administration to androgen-sensitive prostate cancer cells.

    Science.gov (United States)

    Ghashghaei, Maryam; Paliouras, Miltiadis; Heravi, Mitra; Bekerat, Hamed; Trifiro, Mark; Niazi, Tamim M; Muanza, Thierry

    2018-01-01

    Prostate cancer (PCa) is a progressive disease and the most diagnosed cancer in men. The current standard of care for high-risk localized PCa is a combination of androgen deprivation therapy (ADT) and radiation (XRT). The majority of these patients however become resistant due to incomplete responses to ADT as a result of selective cells maintaining androgen receptor (AR) activity. Improvement can be made if increasing radiosensitivity is realized. Therefore, the aim of this study is to investigate the efficacy of the next-generation PCa drug Enzalutamide (ENZA), as a radiosensitizer in XRT therapy. Using a number of androgen-dependent (LNCaP, PC3-T877A) and androgen-independent (C4-2, 22RV1, PC3, PC3-AR V7) cell lines, the effect of ENZA as a radiosensitizer was studied alone or in combination with ADT and/or XRT. Cell viability and cell survival were assessed, along with determination of cell cycle arrest, DNA damage response and repair, apoptosis and senescence. Our results indicated that either ENZA alone (in AR positive, androgen-dependent PCa cells) or in combination with ADT (in AR positive, hormone-insensitive PCa cells) potentiates radiation response [Dose enhancement factor (DEF) of 1.75 in LNCAP and 1.35 in C4-2] stronger than ADT + XRT conditions. Additionally, ENZA sensitized androgen dependent PCa cells to XRT in a schedule-dependent manner, where concurrent administration of ENZA and radiation lead to a maximal radiosensitization when compared to either drug administration prior or after XRT. In LNCaP cells, ENZA treatment significantly prolonged the presence of XRT-induced phospho-γH2AX up to 24 h after treatment; suggesting enhanced DNA damage. It also significantly increased XRT-induced apoptosis and senescence. Our data indicates that ENZA acts as a much stronger radiosensitizer compared to ADT. We have also observed that its efficacy is schedule dependent and related to increased levels of DNA damage and a delay of DNA repair processes

  12. Enhancement of Spike-Timing-Dependent Plasticity in Spiking Neural Systems with Noise.

    Science.gov (United States)

    Nobukawa, Sou; Nishimura, Haruhiko

    2016-08-01

    Synaptic plasticity is widely recognized to support adaptable information processing in the brain. Spike-timing-dependent plasticity, one subtype of plasticity, can lead to synchronous spike propagation with temporal spiking coding information. Recently, it was reported that in a noisy environment, like the actual brain, the spike-timing-dependent plasticity may be made efficient by the effect of stochastic resonance. In the stochastic resonance, the presence of noise helps a nonlinear system in amplifying a weak (under barrier) signal. However, previous studies have ignored the full variety of spiking patterns and many relevant factors in neural dynamics. Thus, in order to prove the physiological possibility for the enhancement of spike-timing-dependent plasticity by stochastic resonance, it is necessary to demonstrate that this stochastic resonance arises in realistic cortical neural systems. In this study, we evaluate this stochastic resonance phenomenon in the realistic cortical neural system described by the Izhikevich neuron model and compare the characteristics of typical spiking patterns of regular spiking, intrinsically bursting and chattering experimentally observed in the cortex.

  13. Dreaming of a learning task is associated with enhanced sleep-dependent memory consolidation.

    Science.gov (United States)

    Wamsley, Erin J; Tucker, Matthew; Payne, Jessica D; Benavides, Joseph A; Stickgold, Robert

    2010-05-11

    It is now well established that postlearning sleep is beneficial for human memory performance. Meanwhile, human and animal studies have demonstrated that learning-related neural activity is re-expressed during posttraining nonrapid eye movement (NREM) sleep. NREM sleep processes appear to be particularly beneficial for hippocampus-dependent forms of memory. These observations suggest that learning triggers the reactivation and reorganization of memory traces during sleep, a systems-level process that in turn enhances behavioral performance. Here, we hypothesized that dreaming about a learning experience during NREM sleep would be associated with improved performance on a hippocampus-dependent spatial memory task. Subjects were trained on a virtual navigation task and then retested on the same task 5 hr after initial training. Improved performance at retest was strongly associated with task-related dream imagery during an intervening afternoon nap. Task-related thoughts during wakefulness, in contrast, did not predict improved performance. These observations suggest that sleep-dependent memory consolidation in humans is facilitated by the offline reactivation of recently formed memories, and furthermore that dream experiences reflect this memory processing. That similar effects were not observed during wakefulness suggests that these mnemonic processes are specific to the sleep state. (c) 2010 Elsevier Ltd. All rights reserved.

  14. Nano-sized Soluplus® polymeric micelles enhance the induction of tetanus toxin neutralising antibody response following transcutaneous immunisation with tetanus toxoid.

    Science.gov (United States)

    Saydam, Manolya; Cheng, Woei Ping; Palmer, Nathan; Tierney, Robert; Francis, Robert; MacLellan-Gibson, Kirsty; Khan, Ambreen; Mawas, Fatme

    2017-04-25

    The use of Soluplus® polymeric micelles as a novel adjuvant for tetanus toxoid (TTxd) in transcutaneous immunisation was evaluated. TTxd was added to Soluplus® polymeric micelles to form TTxd-Soluplus® nano-aggregates with a size of 68nm. Non-adjuvanted TTxd commonly induces very poor antibody response by the transcutaneous route. However, in this study, the use of TTxd-Soluplus® resulted in a significant increase in the antibody response to TTxd, which was similar to that induced in the presence of CPG-oligodeoxynucleotides (CPG-ODNs) adjuvant. The toxin neutralising potency of the immune sera induced by TTxd-Soluplus® was also much stronger than that from TTxd alone, in a passive transfer experiment in mice. Soluplus® also enhanced the immunogenicity of the toxoid when TTxd-Soluplus® was stored at 4°C for 4weeks, but not at higher temperatures. Confocal microscopy imaging showed a much higher uptake of TTxd in the epidermis and dermis layers of the skin when it was associated with Soluplus®, suggesting that the mechanism for Soluplus® adjuvanticity is through enhanced uptake of the TTxd through the skin. Overall, our findings demonstrated that Soluplus® is an effective novel adjuvant for transcutaneous immunisation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Context-dependent enhancer selection confers alternate modes of notch regulation on argos.

    Science.gov (United States)

    Housden, Benjamin E; Terriente-Felix, Ana; Bray, Sarah J

    2014-02-01

    Wiring between signaling pathways differs according to context, as exemplified by interactions between Notch and epidermal growth factor receptor (EGFR) pathways, which are cooperative in some contexts but antagonistic in others. To investigate mechanisms that underlie different modes of cross talk, we have focused on argos, an EGFR pathway regulator in Drosophila melanogaster which is upregulated by Notch in adult muscle progenitors but is repressed in the wing. Results show that the alternate modes of cross talk depend on the engagement of enhancers with opposite regulatory logic, which are selected by context-determining factors. This is likely to be a general mechanism for enabling the wiring between these pathways to switch according to context.

  16. Type i CD20 antibodies recruit the B cell receptor for complement-dependent lysis of malignant B cells

    DEFF Research Database (Denmark)

    Engelberts, P. J.; Voorhorst, M.; Schuurman, J.

    2016-01-01

    . We hypothesized that CD20 Ab-induced clustering of the IgM or IgG BCR was involved in accessory CDC. Indeed, accessory CDC was consistently observed in B cell lines expressing an IgM BCR and in some cell lines expressing an IgG BCR, but it was absent in BCR- B cell lines. A direct relationship...... between BCR expression and accessory CDC was established by transfecting the BCR into CD20+ cells: OFA-F(ab')2 fragments were able to induce CDC in the CD20+BCR+ cell population, but not in the CD20+BCR- population. Importantly, OFA-F(ab')2 fragments were able to induce CDC ex vivo in malignant B cells...... isolated from patients with mantle cell lymphoma and Waldenström macroglobulinemia. In summary, accessory CDC represents a novel effector mechanism that is dependent on type I CD20 Ab-induced BCR clustering. Accessory CDC may contribute to the excellent capacity of type I CD20 Abs to induce CDC...

  17. Atom-Dependent Edge-Enhanced Second-Harmonic Generation on MoS2Monolayers.

    Science.gov (United States)

    Lin, Kuang-I; Ho, Yen-Hung; Liu, Shu-Bai; Ciou, Jian-Jhih; Huang, Bo-Ting; Chen, Christopher; Chang, Han-Ching; Tu, Chien-Liang; Chen, Chang-Hsiao

    2018-02-14

    Edge morphology and lattice orientation of single-crystal molybdenum disulfide (MoS 2 ) monolayers, a transition metal dichalcogenide (TMD), possessing a triangular shape with different edges grown by chemical vapor deposition are characterized by atomic force microscopy and transmission electron microscopy. Multiphoton laser scanning microscopy is utilized to study one-dimensional atomic edges of MoS 2 monolayers with localized midgap electronic states, which result in greatly enhanced optical second-harmonic generation (SHG). Microscopic S-zigzag edge and S-Mo Klein edge (bare Mo atoms protruding from a S-zigzag edge) terminations and the edge-atom dependent resonance energies can therefore be deduced based on SHG images. Theoretical calculations based on density functional theory clearly explain the lower energy of the S-zigzag edge states compared to the corresponding S-Mo Klein edge states. Characterization of the atomic-scale variation of edge-enhanced SHG is a step forward in this full-optical and high-yield technique of atomic-layer TMDs.

  18. Ultrasound-enhanced penetration through sclera depends on frequency of sonication and size of macromolecules.

    Science.gov (United States)

    Chau, Ying; Suen, Wai Leung Langston; Tse, Ho Yan; Wong, Hoi Sang

    2017-03-30

    We previously employed ultrasound as a needleless approach to deliver macromolecules via the transscleral route to the back of the eye in live animals (Suen et al., 2013). Here, we investigated the nature of the ultrasound-enhanced transport through sclera, the outermost barrier in the transscleral route. Thus, the possible role of cavitation from ultrasound was explored; its effect during and after sonication on scleral penetration was measured; and the dependence on the size of macromolecules was determined. We applied ultrasound frequency from 40kHz to 3MHz at I SATA (spatial-average-temporal-average intensity) of 0.05W/cm 2 to fresh rabbit sclera ex vivo. Fluorescent dextran of size 20kDa to 150kDa was used as macromolecular probes. We measured the distance of penetration of the probes through the sclera over 30s during sonication and over 15min after sonication from cryosectioned tissue images. Deeper penetration in the sclera was observed with decreasing frequency. The presence of stable cavitation was further verified by passive acoustic detection. The effect during sonication increased penetration distance up to 20 fold and was limited to macromolecular probes ≤70kDa. The effect post sonication increased penetration distance up to 3 fold and attributed to the improved intrasscleral transport of macromolecules ≥70kDa. Post-sonication enhancement diminished gradually in 3h. As the extent of cavitation increased with decreasing frequency, the trend observed supports the contribution of (stable) cavitation to enhancing transport through sclera. Effect during sonication was attributed to flow associated with acoustic microstreaming. Effect post sonication was attributed to the temporary increase in scleral permeability. Flow-associated effect was more pronounced but only applied to smaller macromolecules. Copyright © 2017. Published by Elsevier B.V.

  19. Bioorthogonal, two-component delivery systems based on antibody and drug-loaded nanocarriers for enhanced internalization of nanotherapeutics.

    Science.gov (United States)

    Hapuarachchige, Sudath; Zhu, Wenlian; Kato, Yoshinori; Artemov, Dmitri

    2014-02-01

    Nanocarriers play an important role in targeted cancer chemotherapy. The optimal nanocarrier delivery system should provide efficient and highly specific recognition of the target cells and rapid internalization of the therapeutic cargo to reduce systemic toxicity as well as to increase the cytotoxicity to cancer cells. To this end, we developed a two-step, two-component targeted delivery system based on antibody and drug-loaded nanocarrier that uses bioorthogonal click reactions for specific internalization of nanotherapeutics. The pretargeting component, anti-HER2 humanized monoclonal antibody, trastuzumab, functionalized with azide groups labels cancer cells that overexpress HER2 surface receptors. The drug carrier component, dibenzylcyclooctyne substituted albumin conjugated with paclitaxel, reacts specifically with the pretargeting component. These two components form cross-linked clusters on the cell surface, which facilitates the internalization of the complex. This strategy demonstrated substantial cellular internalization of clusters consisted of HER2 receptors, modified trastuzumab and paclitaxel-loaded albumin nanocarriers, and subsequent significant cytotoxicity in HER2-positive BT-474 breast cancer cells. Our results show high efficacy of this strategy for targeted nanotherapeutics. We foresee to broaden the applications of this strategy using agents such as radionuclides, toxins, and interfering RNA. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Toll-like receptor activation enhances cell-mediated immunity induced by an antibody vaccine targeting human dendritic cells

    Directory of Open Access Journals (Sweden)

    Berger Marc A

    2007-01-01

    Full Text Available Abstract Previously, we have successfully targeted the mannose receptor (MR expressed on monocyte-derived dendritic cells (DCs using a fully human MR-specific antibody, B11, as a vehicle to deliver whole protein tumor antigens such as the human chorionic gonadotropin hormone (hCGβ. Since MRs play a role in bridging innate immunity with adaptive immunity we have explored several toll-like receptor (TLR-specific ligands that may synergize with MR targeting and be applicable as adjuvants in the clinic. We demonstrate that antigen-specific helper and cytolytic T cells from both healthy donors and cancer patients were effectively primed with B11-hCGβ-treated autologous DCs when a combination of one or several TLR ligands is used. Specifically, concomitant signaling of DCs via TLR3 with dsRNA (poly I:C and DC TLR 7/8 with Resiquimod (R-848, respectively, elicited efficient antigen presentation-mediated by MR-targeting. We demonstrate that MR and TLRs contribute towards maturation and activation of DCs by a mechanism that may be driven by a combination of adjuvant and antibody vaccines that specifically deliver antigenic targets to DCs.

  1. Osmolyte Effects on Monoclonal Antibody Stability and Concentration-Dependent Protein Interactions with Water and Common Osmolytes.

    Science.gov (United States)

    Barnett, Gregory V; Razinkov, Vladimir I; Kerwin, Bruce A; Blake, Steven; Qi, Wei; Curtis, Robin A; Roberts, Christopher J

    2016-04-07

    Preferential interactions of proteins with water and osmolytes play a major role in controlling the thermodynamics of protein solutions. While changes in protein stability and shifts in phase behavior are often reported with the addition of osmolytes, the underlying protein interactions with water and/or osmolytes are typically inferred rather than measured directly. In this work, Kirkwood-Buff integrals for protein-water interactions (G12) and protein-osmolyte interactions (G23) were determined as a function of osmolyte concentration from density measurements of antistreptavidin immunoglobulin gamma-1 (AS-IgG1) in ternary aqueous solutions for a set of common neutral osmolytes: sucrose, trehalose, sorbitol, and poly(ethylene glycol) (PEG). For sucrose and PEG solutions, both protein-water and protein-osmolyte interactions depend strongly on osmolyte concentrations (c3). Strikingly, both osmolytes change from being preferentially excluded to preferentially accumulated with increasing c3. In contrast, sorbitol and trehalose solutions do not show large enough preferential interactions to be detected by densimetry. G12 and G23 values are used to estimate the transfer free energy for native AS-IgG1 (Δμ2N) and compared with existing models. AS-IgG1 unfolding via calorimetry shows a linear increase in midpoint temperatures as a function of trehalose, sucrose, and sorbitol concentrations, but the opposite behavior for PEG. Together, the results highlight limitations of existing models and common assumptions regarding the mechanisms of protein stabilization by osmolytes. Finally, PEG preferential interactions destabilize the Fab regions of AS-IgG1 more so than the CH2 or CH3 domains, illustrating preferential interactions can be specific to different protein domains.

  2. [A web information system for enhancing management and improving special care services provided to dependent persons].

    Science.gov (United States)

    Alvarez-Bermejo, J A; Hernández-Capel, D M; Belmonte-Ureña, L J; Roca-Piera, J

    2009-01-01

    Ensuring the quality of services provided in centres where dependent persons are seen by specialist services, by improving and enhancing how information -salary, control of tasks, patients' records, etc.- is shared between staff and carers. A web information system has been developed and experimentally deployed to accomplish this. The accuracy of the system was evaluated by assessing how confident the employees were with it rather than relying on statistical data. It was experimentally deployed since January 2009 in Asociación de Personas con Discapacidad "El Saliente" that manages several day centres in Almeria, for dependent persons over 65 years old, particularly those affected by Alzheimer' disease. Incidence data was collected during the experimental period. A total of 84% of the employees thought that the system helped to manage documents, administrative duties, etc., and 92.4% said they could attend to really important tasks because the system was responsible for alerting them of every task, such as medication timetables, checking all patients were present (to prevent an Alzheimer affected person leaving the centre) etc. During this period the incidences reported were reduced by about a 30%, although data is still partially representative. As the life expectancy of the population gets longer, these centres will increase. Providing systems such as the one presented here would be of great help for administrative duties (sensitive data protection...) as well as ensuring high quality care and attention.

  3. Bromodomain Protein BRD4 Is Required for Estrogen Receptor-Dependent Enhancer Activation and Gene Transcription

    Directory of Open Access Journals (Sweden)

    Sankari Nagarajan

    2014-07-01

    Full Text Available The estrogen receptor α (ERα controls cell proliferation and tumorigenesis by recruiting various cofactors to estrogen response elements (EREs to control gene transcription. A deeper understanding of these transcriptional mechanisms may uncover therapeutic targets for ERα-dependent cancers. We show that BRD4 regulates ERα-induced gene expression by affecting elongation-associated phosphorylation of RNA polymerase II (RNAPII and histone H2B monoubiquitination. Consistently, BRD4 activity is required for proliferation of ER+ breast and endometrial cancer cells and uterine growth in mice. Genome-wide studies revealed an enrichment of BRD4 on transcriptional start sites of active genes and a requirement of BRD4 for H2B monoubiquitination in the transcribed region of estrogen-responsive genes. Importantly, we demonstrate that BRD4 occupancy on distal EREs enriched for H3K27ac is required for recruitment and elongation of RNAPII on EREs and the production of ERα-dependent enhancer RNAs. These results uncover BRD4 as a central regulator of ERα function and potential therapeutic target.

  4. Ghrelin enhances food intake and carbohydrate oxidation in a nitric oxide dependent manner.

    Science.gov (United States)

    Abtahi, Shayan; Mirza, Aaisha; Howell, Erin; Currie, Paul J

    2017-09-01

    In the present study we sought to investigate interactions between hypothalamic nitric oxide (NO) and ghrelin signaling on food intake and energy substrate utilization as measured by the respiratory exchange ratio (RER). Guide cannulae were unilaterally implanted in either the arcuate (ArcN) or paraventricular (PVN) nuclei of male Sprague-Dawley rats. Animals were pretreated with subcutaneous (2.5-10mg/kg/ml) or central (0-100pmol) N-nitro-l-Arginine methyl ester (l-NAME) followed by 50pmol of ghrelin administered into either the ArcN or PVN. Both l-NAME and ghrelin were microinjected at the onset of the active cycle and food intake and RER were assessed 2h postinjection. RER was measured as the ratio of the volume of carbon dioxide expelled relative to the volume of oxygen consumed (VCO 2 /VO 2 ) using an open-circuit indirect calorimeter. Our results demonstrated that peripheral and central l-NAME pretreatment dose-dependently attenuated ghrelin induced increases in food intake and RER in either the ArcN or PVN. In fact the 100pmol dose largely reversed the metabolic effects of ghrelin in both anatomical regions. These findings suggest that ghrelin enhancement of food intake and carbohydrate oxidation in the rat ArcN and PVN is NO-dependent. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Annealing time dependent up-conversion luminescence enhancement in magnesium–tellurite glass

    Energy Technology Data Exchange (ETDEWEB)

    Amjad, Raja J., E-mail: rajajunaid25@gmail.com [Advanced Optical Material Research Group, Faculty of Science, Universiti Teknologi Malaysia, Skudai, Johor bahru 81310 (Malaysia); Centre for Solid State Physics, University of the Punjab, QAC, Lahore 54590 (Pakistan); Sahar, M.R.; Ghoshal, S.K.; Dousti, M.R. [Advanced Optical Material Research Group, Faculty of Science, Universiti Teknologi Malaysia, Skudai, Johor bahru 81310 (Malaysia); Riaz, S. [Centre for Solid State Physics, University of the Punjab, QAC, Lahore 54590 (Pakistan); Samavati, A.R.; Arifin, R. [Advanced Optical Material Research Group, Faculty of Science, Universiti Teknologi Malaysia, Skudai, Johor bahru 81310 (Malaysia); Naseem, S. [Centre for Solid State Physics, University of the Punjab, QAC, Lahore 54590 (Pakistan)

    2013-04-15

    Silver nanoparticles (NPs) embedded Er{sup 3+} ions doped magnesium–tellurite glasses are prepared using melt quenching technique. Heat treatment with different time intervals above the glass transition temperature is applied in order to reduce the silver ions (Ag{sup +}) to silver NPs (Ag{sup o}). The transmission electron microscopy (TEM), differential thermal analyses (DTA), UV–vis-NIR absorption spectroscopy and photoluminescence (PL) spectroscopy are used to examine annealing time dependent structural and optical properties. The characteristics temperatures such as glass transition temperature (T{sub g}), crystallization temperature (T{sub c}) and melting temperature (T{sub m}) obtained from DTA for an as prepared sample are 322 °C, 450 °C and 580 °C, respectively. TEM image clearly shows the homogeneous distribution of silver NPs with an average diameter ∼12 nm. The observed localized surface plasmon resonance (LSPR) band is evidenced at 534 nm. Furthermore, the infrared to visible frequency up-conversion (UC) emission under 786 nm excitation exhibits three emission bands centered at 532 nm, 554 nm and 634 nm corresponding to {sup 2}H{sub 11/2}→{sup 4}I{sub 15/2}, {sup 4}S{sub 3/2}→{sup 4}I{sub 15/2} and {sup 4}F{sub 9/2}→{sup 4}I{sub 15/2} transitions of Er{sup 3+}, respectively. Intensity of all the bands is found to enhance by increasing the annealing time up to 24 h. However, further increase in the annealing time duration (∼40 h) reduces the intensity. Enhancement in the luminescence intensity is understood in terms of the local field effect of the silver NPs whereas the quenching is attributed to the energy transfer from Er{sup 3+} ions to silver NPs. -- Highlights: ► Er{sup 3+}-doped silver NPs embedded magnesium–tellurite glasses are prepared. ► TEM confirms the successful precipitation of spherical NPs by heat treatment (HT). ► Luminescence is enhanced due to the growth of NPs after HT up to 24 h. ► With HT>24 h (40 h

  6. Flow cytometric readout based on Mitotracker Red CMXRos staining of live asexual blood stage malarial parasites reliably assesses antibody dependent cellular inhibition

    Directory of Open Access Journals (Sweden)

    Jogdand Prajakta S

    2012-07-01

    Full Text Available Abstract Background Functional in vitro assays could provide insights into the efficacy of malaria vaccine candidates. For estimating the anti-parasite effect induced by a vaccine candidate, an accurate determination of live parasite count is an essential component of most in vitro bioassays. Although traditionally parasites are counted microscopically, a faster, more accurate and less subjective method for counting parasites is desirable. In this study mitochondrial dye (Mitotracker Red CMXRos was used for obtaining reliable live parasite counts through flow cytometry. Methods Both asynchronous and tightly synchronized asexual blood stage cultures of Plasmodium falciparum were stained with CMXRos and subjected to detection by flow cytometry and fluorescence microscopy. The parasite counts obtained by flow cytometry were compared to standard microscopic counts obtained through examination of Giemsa-stained thin smears. A comparison of the ability of CMXRos to stain live and compromised parasites (induced by either medium starvation or by anti-malarial drug treatment was carried out. Finally, parasite counts obtained by CMXRos staining through flow cytometry were used to determine specific growth inhibition index (SGI in an antibody-dependent cellular inhibition (ADCI assay. Results Mitotracker Red CMXRos can reliably detect live intra-erythrocytic stages of P. falciparum. Comparison between staining of live with compromised parasites shows that CMXRos predominantly stains live parasites with functional mitochondria. Parasite counts obtained by CMXRos staining and flow cytometry were highly reproducible and can reliably determine the ability of IgG from hyper-immune individuals to inhibit parasite growth in presence of monocytes in ADCI assay. Further, a dose-dependent parasite growth inhibitory effect could be detected for both total IgG purified from hyper-immune sera and affinity purified IgGs against the N-terminal non-repeat region of GLURP

  7. Flow cytometric readout based on Mitotracker Red CMXRos staining of live asexual blood stage malarial parasites reliably assesses antibody dependent cellular inhibition.

    Science.gov (United States)

    Jogdand, Prajakta S; Singh, Susheel K; Christiansen, Michael; Dziegiel, Morten H; Singh, Subhash; Theisen, Michael

    2012-07-20

    Functional in vitro assays could provide insights into the efficacy of malaria vaccine candidates. For estimating the anti-parasite effect induced by a vaccine candidate, an accurate determination of live parasite count is an essential component of most in vitro bioassays. Although traditionally parasites are counted microscopically, a faster, more accurate and less subjective method for counting parasites is desirable. In this study mitochondrial dye (Mitotracker Red CMXRos) was used for obtaining reliable live parasite counts through flow cytometry. Both asynchronous and tightly synchronized asexual blood stage cultures of Plasmodium falciparum were stained with CMXRos and subjected to detection by flow cytometry and fluorescence microscopy. The parasite counts obtained by flow cytometry were compared to standard microscopic counts obtained through examination of Giemsa-stained thin smears. A comparison of the ability of CMXRos to stain live and compromised parasites (induced by either medium starvation or by anti-malarial drug treatment) was carried out. Finally, parasite counts obtained by CMXRos staining through flow cytometry were used to determine specific growth inhibition index (SGI) in an antibody-dependent cellular inhibition (ADCI) assay. Mitotracker Red CMXRos can reliably detect live intra-erythrocytic stages of P. falciparum. Comparison between staining of live with compromised parasites shows that CMXRos predominantly stains live parasites with functional mitochondria. Parasite counts obtained by CMXRos staining and flow cytometry were highly reproducible and can reliably determine the ability of IgG from hyper-immune individuals to inhibit parasite growth in presence of monocytes in ADCI assay. Further, a dose-dependent parasite growth inhibitory effect could be detected for both total IgG purified from hyper-immune sera and affinity purified IgGs against the N-terminal non-repeat region of GLURP in ADCI assays coupled with determination of

  8. Low-dose synergistic immunosuppression of T-dependent antibody responses by polycyclic aromatic hydrocarbons and arsenic in C57BL/6J murine spleen cells

    International Nuclear Information System (INIS)

    Li Qian; Lauer, Fredine T.; Liu Kejian; Hudson, Laurie G.; Burchiel, Scott W.

    2010-01-01

    Polycyclic aromatic hydrocarbons (PAHs) and arsenic are both environmental agents that are known to have significant immunotoxicity. Previous studies have shown that PAH exposure of spleen cells in vitro produces significant immune suppression of humoral immunity, especially when P450 activation products are examined. Exposure to arsenic, particularly sodium arsenite, has also been found to be suppressive to antibody responses in vitro and in vivo. The purpose of the present studies was to examine the immunotoxicity of PAHs and arsenite following coexposures with the theory being that the agents may exert synergistic actions, which might be based on their different mechanisms of action. Spleen cells were isolated from male C57BL/6J wild-type mice and treated with PAHs and/or arsenic (arsenite or arsenate). Immunotoxicity assays were used to assess the T-dependent antibody response (TDAR) to sheep red blood cells (SRBCs), measured by a direct plaque-forming cell (PFC) assay. Cell viability was measured by trypan blue staining. Spleen cell viability was not altered following 4 days of PAH and/or arsenic treatment. However, the TDAR demonstrated suppression by both PAHs and arsenic in a concentration-dependent manner. p53 was also induced by NaAsO 2 (As 3+ ) and PAHs alone or in combination. The PAHs and their metabolites investigated included benzo[a]pyrene (BaP), BaP-7,8-diol, BaP-7,8-diol-9,10-epoxide (BPDE), 7,12-dimethylbenz[a]anthracene (DMBA), DMBA-3,4-diol, dibenzo[a,l]pyrene (DB[a,l]P). PAH metabolites were found to be more potent than parent compounds in producing immunosuppression and inducing p53 expression. Interestingly, DB[a,l]P, a potent carcinogenic PAH not previously characterized for immunotoxicity, was also found to be strongly immunosuppressive. Arsenite (NaAsO 2 , As 3+ ) was found to produce immunosuppression at concentrations as low as 0.5 μM and was immunosuppressive at a 10-fold lower concentration than sodium arsenate (Na 2 HAsO 4 , As 5

  9. CD14-dependent monocyte isolation enhances phagocytosis of listeria monocytogenes by proinflammatory, GM-CSF-derived macrophages.

    Directory of Open Access Journals (Sweden)

    Caroline Neu

    Full Text Available Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF or macrophage colony-stimulating factor (M-CSF into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ stained positive for CD206 and M-CSF-derived macrophages (M-Mφ for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most

  10. Antibody-mediated delivery of interleukin-2 to the stroma of breast cancer strongly enhances the potency of chemotherapy.

    Science.gov (United States)

    Mårlind, Jessica; Kaspar, Manuela; Trachsel, Eveline; Sommavilla, Roberto; Hindle, Stuart; Bacci, Camilla; Giovannoni, Leonardo; Neri, Dario

    2008-10-15

    There is an interest in the discovery of biopharmaceuticals, which are well tolerated and which potentiate the action of anthracyclines and taxanes in breast cancer therapy. We have produced a recombinant fusion protein, composed of the human antibody fragment scFv(F16) fused to human interleukin-2 (F16-IL2), and tested its therapeutic performance in the MDA-MB-231 xenograft model of human breast cancer. The F16 antibody is specific to the alternatively spliced A1 domain of tenascin-C, which is virtually undetectable in normal tissues but is strongly expressed in the neovasculature and stroma of breast cancer. When used as monotherapy, F16-IL2 displayed a strikingly superior therapeutic benefit compared with unconjugated recombinant IL-2. The administration of doxorubicin either before (8 days, 24 h, or 2 h) or simultaneously with the injection of F16-IL2 did not decrease the accumulation of immunocytokine in the tumor as measured by quantitative biodistribution analysis. Therapy experiments, featuring five once per week coadministrations of 20 mug F16-IL2 and doxorubicin, showed a statistically significant reduction of tumor growth rate and prolongation of survival at a 4 mg/kg doxorubicin dose but not at a 1 mg/kg dose. By contrast, combination of F16-IL2 with paclitaxel (5 and 1 mg/kg) exhibited a significant therapeutic benefit compared with paclitaxel alone at both dose levels. F16-IL2, alone or in combination with doxorubicin, was well tolerated in cynomolgus monkeys at doses equivalent to the ones now used in clinical studies. F16-IL2 may represent a new useful biopharmaceutical for the treatment of breast cancer.

  11. Enhanced attenuation of meningeal inflammation and brain edema by concomitant administration of anti-CD18 monoclonal antibodies and dexamethasone in experimental Haemophilus meningitis.

    Science.gov (United States)

    Sáez-Llorens, X; Jafari, H S; Severien, C; Parras, F; Olsen, K D; Hansen, E J; Singer, I I; McCracken, G H

    1991-12-01

    Antiinflammatory therapy has been shown to reduce the adverse pathophysiological consequences that occur in bacterial meningitis and to improve outcome from disease. In the present study, modulation of two principal steps of the meningeal inflammatory cascade was accomplished by concomitant administration of dexamethasone to diminish overproduction of cytokines in response to a bacterial stimulus and of a monoclonal antibody directed against adhesion-promoting receptors on leukocytes to inhibit recruitment of white blood cells into the subarachnoid space. Dexamethasone and antibody therapy produced a marked attenuation of all indices of meningeal inflammation and reduction of brain water accumulation after H. influenzae-induced meningitis in rabbits compared with results of each agent given alone and of untreated animals. In addition, the enhanced host's meningeal inflammatory reaction that follows antibiotic-induced bacterial lysis was profoundly ameliorated when dual therapy was administered without affecting clearance rates of bacteria from cerebrospinal fluid and vascular compartments. The combination of both therapeutic approaches may offer a promising mode of treatment to improve further the outcome from bacterial meningitis.

  12. Age-dependent association between IgG2 and IgG3 subclasses to Pf332-C231 antigen and protection from malaria, and induction of protective antibodies by sub-patent malaria infections, in Daraweesh

    DEFF Research Database (Denmark)

    Giha, Hayder A; Nasr, Amre; Iriemenam, Nnaemeka C

    2010-01-01

    , three variable markers for protection were emerged, two age-dependent, the antibody response to Pf332-C231 and an unidentified marker (likely immune response to other antigens), and the third was an age-independent unidentified marker (possibly gene polymorphisms). In conclusion, this report suggests...

  13. First Phase I human clinical trial of a killed whole-HIV-1 vaccine: demonstration of its safety and enhancement of anti-HIV antibody responses.

    Science.gov (United States)

    Choi, Eunsil; Michalski, Chad J; Choo, Seung Ho; Kim, Gyoung Nyoun; Banasikowska, Elizabeth; Lee, Sangkyun; Wu, Kunyu; An, Hwa-Yong; Mills, Anthony; Schneider, Stefan; Bredeek, U Fritz; Coulston, Daniel R; Ding, Shilei; Finzi, Andrés; Tian, Meijuan; Klein, Katja; Arts, Eric J; Mann, Jamie F S; Gao, Yong; Kang, C Yong

    2016-11-28

    Vaccination with inactivated (killed) whole-virus particles has been used to prevent a wide range of viral diseases. However, for an HIV vaccine this approach has been largely negated due to inherent safety concerns, despite the ability of killed whole-virus vaccines to generate a strong, predominantly antibody-mediated immune response in vivo. HIV-1 Clade B NL4-3 was genetically modified by deleting the nef and vpu genes and substituting the coding sequence for the Env signal peptide with that of honeybee melittin signal peptide to produce a less virulent and more replication efficient virus. This genetically modified virus (gmHIV-1 NL4-3 ) was inactivated and formulated as a killed whole-HIV vaccine, and then used for a Phase I human clinical trial (Trial Registration: Clinical Trials NCT01546818). The gmHIV-1 NL4-3 was propagated in the A3.01 human T cell line followed by virus purification and inactivation with aldrithiol-2 and γ-irradiation. Thirty-three HIV-1 positive volunteers receiving cART were recruited for this observer-blinded, placebo-controlled Phase I human clinical trial to assess the safety and immunogenicity. Genetically modified and killed whole-HIV-1 vaccine, SAV001, was well tolerated with no serious adverse events. HIV-1 NL4-3 -specific PCR showed neither evidence of vaccine virus replication in the vaccine virus-infected human T lymphocytes in vitro nor in the participating volunteers receiving SAV001 vaccine. Furthermore, SAV001 with adjuvant significantly increased the pre-existing antibody response to HIV-1 proteins. Antibodies in the plasma of vaccinees were also found to recognize HIV-1 envelope protein on the surface of infected cells as well as showing an enhancement of broadly neutralizing antibodies inhibiting tier I and II of HIV-1 B, D, and A subtypes. The killed whole-HIV vaccine, SAV001, is safe and triggers anti-HIV immune responses. It remains to be determined through an appropriate trial whether this immune response prevents

  14. PPARδ activation acts cooperatively with 3-phosphoinositide-dependent protein kinase-1 to enhance mammary tumorigenesis.

    Directory of Open Access Journals (Sweden)

    Claire B Pollock

    Full Text Available Peroxisome proliferator-activated receptorδ (PPARδ is a transcription factor that is associated with metabolic gene regulation and inflammation. It has been implicated in tumor promotion and in the regulation of 3-phosphoinositide-dependent kinase-1 (PDK1. PDK1 is a key regulator of the AGC protein kinase family, which includes the proto-oncogene AKT/PKB implicated in several malignancies, including breast cancer. To assess the role of PDK1 in mammary tumorigenesis and its interaction with PPARδ, transgenic mice were generated in which PDK1 was expressed in mammary epithelium under the control of the MMTV enhancer/promoter region. Transgene expression increased pT308AKT and pS9GSK3β, but did not alter phosphorylation of mTOR, 4EBP1, ribosomal protein S6 and PKCα. The transgenic mammary gland also expressed higher levels of PPARδ and a gene expression profile resembling wild-type mice maintained on a diet containing the PPARδ agonist, GW501516. Both wild-type and transgenic mice treated with GW501516 exhibited accelerated rates of tumor formation that were more pronounced in transgenic animals. GW501516 treatment was accompanied by a distinct metabolic gene expression and metabolomic signature that was not present in untreated animals. GW501516-treated transgenic mice expressed higher levels of fatty acid and phospholipid metabolites than treated wild-type mice, suggesting the involvement of PDK1 in enhancing PPARδ-driven energy metabolism. These results reveal that PPARδ activation elicits a distinct metabolic and metabolomic profile in tumors that is in part related to PDK1 and AKT signaling.

  15. Extracellular Ca(2+)-dependent enhancement of cytocidal potency of zoledronic acid in human oral cancer cells.

    Science.gov (United States)

    Inoue, Sayaka; Arai, Naoya; Tomihara, Kei; Takashina, Michinori; Hattori, Yuichi; Noguchi, Makoto

    2015-08-15

    Direct antitumor effects of bisphosphonates (BPs) have been demonstrated in various cancer cells in vitro. However, the effective concentrations of BPs are typically much higher than their clinically relevant concentrations. Oral cancers frequently invade jawbone and may lead to the release of Ca(2+) in primary lesions. We investigated the effects of the combined application of zoledronic acid (ZA) and Ca(2+) on proliferation and apoptosis of oral cancer cells. Human oral cancer cells, breast cancer cells, and colon cancer cells were treated with ZA at a wide range of concentrations in different Ca(2+) concentration environments. Under a standard Ca(2+) concentration (0.6mM), micromolar concentrations of ZA were required to inhibit oral cancer cell proliferation. Increasing extracellular Ca(2+) concentrations greatly enhanced the potency of the ZA cytocidal effect. The ability of Ca(2+) to enhance the cytocidal effects of ZA was negated by the Ca(2+)-selective chelator EGTA. In contrast, the cytocidal effect of ZA was less pronounced in breast and colon cancer cells regardless of whether extracellular Ca(2+) was elevated. In oral cancer cells incubated with 1.6mM Ca(2+), ZA up-regulated mitochondrial Bax expression and increased mitochondrial Ca(2+) uptake. This was associated with decreased mitochondrial membrane potential and increased release of cytochrome c. We suggest that ZA can specifically produce potent cytocidal activity in oral cancer cells in an extracellular Ca(2+)-dependent manner, implying that BPs may be useful for treatment of oral squamous cell carcinoma with jawbone invasion leading to the hypercalcemic state. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Transferred nuclear Overhauser enhancement experiments show that the monoclonal antibody strep 9 selects a local minimum conformation of a Streptococcus group A trisaccharide-hapten.

    Science.gov (United States)

    Weimar, T; Harris, S L; Pitner, J B; Bock, K; Pinto, B M

    1995-10-17

    Transferred nuclear Overhauser enhancement (TRNOE) experiments have been performed to investigate the bound conformation of the trisaccharide repeating unit of the Streptococcus Group A cell-wall polysaccharide. Thus, the conformations of propyl 3-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2-O-(alpha-L-rhamnopyran osyl)- alpha-L-rhamnopyranoside [C(A')B] (1) as a free ligand and when complexed to the monoclonal antibody Strep 9 were examined. Improved insights about the conformational preferences of the glycosidic linkages of the trisaccharide ligand showed that the free ligand populates various conformations in aqueous solution, thus displaying relatively flexible behavior. The NOE HNAc-H2A', which was not detected in previous work, accounts for a conformation at the beta-(1-->3) linkage with a phi angle of approximately 180 degrees. Observed TRNOEs for the complex are weak, and their analysis was further complicated by spin diffusion. With the use of transferred rotating-frame Overhauser enhancement (TRROE) experiments, the amount of spin diffusion was assessed experimentally, proving that all of the observed long-range TRNOEs arose through spin diffusion. Four interglycosidic distances, derived from the remaining TRNOEs and TRROEs, together with repulsive constraints, derived from the absence of TRROE effects, were used as input parameters in simulated annealing and molecular mechanics calculations to determine the bound conformation of the trisaccharide. Complexation by the antibody results in the selection of one defined conformation of the carbohydrate hapten. This bound conformation, which is a local energy minimum on the energy maps calculated for the trisaccharide ligand, shows only a change from a +gauche to a -gauche orientation at the psi angle of the alpha-(1-->2) linkage when compared to the global minimum conformation. The results infer that the bound conformation of the Streptococcus Group A cell-wall polysaccharide is different from its

  17. The NS1 Glycoprotein Can Generate Dramatic Antibody-Enhanced Dengue Viral Replication in Normal Out-Bred Mice Resulting in Lethal Multi-Organ Disease

    Science.gov (United States)

    Falconar, Andrew K. I.; Martinez, Fernando

    2011-01-01

    Antibody-enhanced replication (AER) of dengue type-2 virus (DENV-2) strains and production of antibody-enhanced disease (AED) was tested in out-bred mice. Polyclonal antibodies (PAbs) generated against the nonstructural-1 (NS1) glycoprotein candidate vaccine of the New Guinea-C (NG-C) or NSx strains reacted strongly and weakly with these antigens, respectively. These PAbs contained the IgG2a subclass, which cross-reacted with the virion-associated envelope (E) glycoprotein of the DENV-2 NSx strain, suggesting that they could generate its AER via all mouse Fcγ-receptor classes. Indeed, when these mice were challenged with a low dose (AER/AED. These AER/AED mice developed life-threatening acute respiratory distress syndrome (ARDS), displayed by diffuse alveolar damage (DAD) resulting from i) dramatic interstitial alveolar septa-thickening with mononuclear cells, ii) some hyperplasia of alveolar type-II pneumocytes, iii) copious intra-alveolar protein secretion, iv) some hyaline membrane-covered alveolar walls, and v) DENV-2 antigen-positive alveolar macrophages. These mice also developed meningo-encephalitis, with greater than 90,000-fold DENV-2 AER titers in microglial cells located throughout their brain parenchyma, some of which formed nodules around dead neurons. Their spleens contained infiltrated megakaryocytes with DENV-2 antigen-positive red-pulp macrophages, while their livers displayed extensive necrosis, apoptosis and macro- and micro-steatosis, with DENV-2 antigen-positive Kuppfer cells and hepatocytes. Their infections were confirmed by DENV-2 isolations from their lungs, spleens and livers. These findings accord with those reported in fatal human “severe dengue” cases. This DENV-2 AER/AED was blocked by high concentrations of only the NG-C NS1 glycoprotein. These results imply a potential hazard of DENV NS1 glycoprotein-based vaccines, particularly against DENV strains that contain multiple mutations or genetic recombination within or between their

  18. The NS1 glycoprotein can generate dramatic antibody-enhanced dengue viral replication in normal out-bred mice resulting in lethal multi-organ disease.

    Directory of Open Access Journals (Sweden)

    Andrew K I Falconar

    Full Text Available Antibody-enhanced replication (AER of dengue type-2 virus (DENV-2 strains and production of antibody-enhanced disease (AED was tested in out-bred mice. Polyclonal antibodies (PAbs generated against the nonstructural-1 (NS1 glycoprotein candidate vaccine of the New Guinea-C (NG-C or NSx strains reacted strongly and weakly with these antigens, respectively. These PAbs contained the IgG2a subclass, which cross-reacted with the virion-associated envelope (E glycoprotein of the DENV-2 NSx strain, suggesting that they could generate its AER via all mouse Fcγ-receptor classes. Indeed, when these mice were challenged with a low dose (<0.5 LD₅₀ of the DENV-2 NSx strain, but not the NG-C strain, they all generated dramatic and lethal DENV-2 AER/AED. These AER/AED mice developed life-threatening acute respiratory distress syndrome (ARDS, displayed by diffuse alveolar damage (DAD resulting from i dramatic interstitial alveolar septa-thickening with mononuclear cells, ii some hyperplasia of alveolar type-II pneumocytes, iii copious intra-alveolar protein secretion, iv some hyaline membrane-covered alveolar walls, and v DENV-2 antigen-positive alveolar macrophages. These mice also developed meningo-encephalitis, with greater than 90,000-fold DENV-2 AER titers in microglial cells located throughout their brain parenchyma, some of which formed nodules around dead neurons. Their spleens contained infiltrated megakaryocytes with DENV-2 antigen-positive red-pulp macrophages, while their livers displayed extensive necrosis, apoptosis and macro- and micro-steatosis, with DENV-2 antigen-positive Kuppfer cells and hepatocytes. Their infections were confirmed by DENV-2 isolations from their lungs, spleens and livers. These findings accord with those reported in fatal human "severe dengue" cases. This DENV-2 AER/AED was blocked by high concentrations of only the NG-C NS1 glycoprotein. These results imply a potential hazard of DENV NS1 glycoprotein-based vaccines

  19. High antibody-dependent cellular cytotoxicity responses are correlated with strong CD8 T cell viral suppressive activity but not with B57 status in HIV-1 elite controllers.

    Directory of Open Access Journals (Sweden)

    Olivier Lambotte

    Full Text Available The role of Antibody-dependent cellular cytotoxicity (ADCC responses in HIV-1 controllers is still unclear due to the heterogeneity of these patients. We analyzed 67 HIV-1 controllers and found significantly higher levels of ADCC antibodies in controllers versus viremic subjects (p = 0.017. Moreover, multivariate analysis revealed significantly higher ADCC titers in HLA B57- controllers compared to HLA-B57+ ones (p = 0.0086. These data suggest a role for ADCC in immune control of HIV, especially in HLA B57 negative controllers.

  20. Enhanced heat transfer is dependent on thickness of graphene films: the heat dissipation during boiling

    Science.gov (United States)

    Ahn, Ho Seon; Kim, Jin Man; Kim, TaeJoo; Park, Su Cheong; Kim, Ji Min; Park, Youngjae; Yu, Dong In; Hwang, Kyoung Won; Jo, HangJin; Park, Hyun Sun; Kim, Hyungdae; Kim, Moo Hwan

    2014-01-01

    Boiling heat transfer (BHT) is a particularly efficient heat transport method because of the latent heat associated with the process. However, the efficiency of BHT decreases significantly with increasing wall temperature when the critical heat flux (CHF) is reached. Graphene has received much recent research attention for applications in thermal engineering due to its large thermal conductivity. In this study, graphene films of various thicknesses were deposited on a heated surface, and enhancements of BHT and CHF were investigated via pool-boiling experiments. In contrast to the well-known surface effects, including improved wettability and liquid spreading due to micron- and nanometer-scale structures, nanometer-scale folded edges of graphene films provided a clue of BHT improvement and only the thermal conductivity of the graphene layer could explain the dependence of the CHF on the thickness. The large thermal conductivity of the graphene films inhibited the formation of hot spots, thereby increasing the CHF. Finally, the provided empirical model could be suitable for prediction of CHF. PMID:25182076

  1. Plasmon resonance enhanced temperature-dependent photoluminescence of Si-V centers in diamond

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Shaoheng [State Key Laboratory of Superhard Materials, Jilin University, Changchun 130012 (China); State Key Laboratory on Integrated Optoelectronics, College of Electronic Science and Engineering, Jilin University, Changchun 130012 (China); Song, Jie; Wang, Qiliang; Liu, Junsong; Li, Hongdong, E-mail: hdli@jlu.edu.cn [State Key Laboratory of Superhard Materials, Jilin University, Changchun 130012 (China); Zhang, Baolin [State Key Laboratory on Integrated Optoelectronics, College of Electronic Science and Engineering, Jilin University, Changchun 130012 (China)

    2015-11-23

    Temperature dependent optical property of diamond has been considered as a very important factor for realizing high performance diamond-based optoelectronic devices. The photoluminescence feature of the zero phonon line of silicon-vacancy (Si-V) centers in Si-doped chemical vapor deposited single crystal diamond (SCD) with localized surface plasmon resonance (LSPR) induced by gold nanoparticles has been studied at temperatures ranging from liquid nitrogen temperature to 473 K, as compared with that of the SCD counterpart in absence of the LSPR. It is found that with LSPR the emission intensities of Si-V centers are significantly enhanced by factors of tens and the magnitudes of the redshift (width) of the emissions become smaller (narrower), in comparison with those of normal emissions without plasmon resonance. More interestingly, these strong Si-V emissions appear remarkably at temperatures up to 473 K, while the spectral feature was not reported in previous studies on the intrinsic Si-doped diamonds when temperatures are higher than room temperature. These findings would lead to reaching high performance diamond-based devices, such as single photon emitter, quantum cryptography, biomarker, and so forth, working under high temperature conditions.

  2. Enhanced field-dependent conductivity of magnetorheological gels with low-doped carbon nanotubes

    Science.gov (United States)

    Qu, Hang; Yu, Miao; Fu, Jie; Yang, Pingan; Liu, Yuxuan

    2017-10-01

    Magnetorheological gels (MRG) exhibit field-dependent conductivity and controllable mechanical properties. In order to extend their application field, filling a large number of traditional conductive materials is the most common means to enhance the poor conductivity of MRG. In this study, the conductivity of MRG is improved by low-doped carbon nanotubes (CNTs). The influence of CNTs on the magnetoresistance of MRG is discussed from two aspects—the improvement in electrical conductivity and the magnetic sensitivity of conductivity variation. The percolation threshold of CNTs in MRG should be between 1 wt% and 2 wt%. The conductivity of a 4 wt% CNT-doped sample increases more than 28 000 times compared with pure MRG. However, there is a cliff-like drop for the range and rate of conductivity variation when the doping amount of CNTs is between 3 wt% and 4 wt%. Therefore, it is concluded that the optimal mass fraction of CNTs is 3%, which can maintain a suitable variation range and a strong conductivity. Compared with pure MRG, its conductivity increases by at least two orders of magnitude. Finally, a sketch of particle motion simulation is developed to understand the improving mechanism and the effect of CNTs.

  3. Capacity Enhancement of Few-Mode Fiber Transmission Systems Impaired by Mode-Dependent Loss

    Directory of Open Access Journals (Sweden)

    El-Mehdi Amhoud

    2018-02-01

    Full Text Available Space-division multiplexing over few-mode fibers is a promising solution to increase the capacity of the future generation of optical transmission systems. Mode-dependent loss (MDL is known to have a detrimental impact on the capacity of few-mode fiber systems. In the presence of MDL, spatial modes experience different attenuations which results in capacity reduction. In this work, we propose a digital signal processing solution and an optical solution to mitigate the impact of MDL and improve the channel capacity. First, we show that statistical channel state information can be used for a better power allocation for spatial modes instead of equal launch power to increase the system capacity. Afterwards, we propose a deterministic mode scrambling strategy to efficiently reduces the impact of MDL and improves few-mode fiber systems capacity. This scrambling strategy can be efficiently combined with the optimal power allocation to further enhance the capacity. Through numerical simulations of the average and outage capacities, we show that the proposed techniques bring significant capacity gains.

  4. Combinatorial treatment with lithium chloride enhances recombinant antibody production in transiently transfected CHO and HEK293E cells

    DEFF Research Database (Denmark)

    Kim, Che Lin; Kwang Ha, Tae; Min Lee, Gyun

    2016-01-01

    Cl was added to the CHO-NK and human embryonic kidney 293E (HEK293E) cell cultures before and/or after transfection with polyethylenimine as a transfection reagent. The effect of this addition on transfection efficiency (pre-treatment) and qp enhancement during TGE (post-treatment) was examined. For the TGE...

  5. Enhanced process understanding and multivariate prediction of the relationship between cell culture process and monoclonal antibody quality.

    Science.gov (United States)

    Sokolov, Michael; Ritscher, Jonathan; MacKinnon, Nicola; Souquet, Jonathan; Broly, Hervé; Morbidelli, Massimo; Butté, Alessandro

    2017-09-01

    This work investigates the insights and understanding which can be deduced from predictive process models for the product quality of a monoclonal antibody based on designed high-throughput cell culture experiments performed at milliliter (ambr-15 ® ) scale. The investigated process conditions include various media supplements as well as pH and temperature shifts applied during the process. First, principal component analysis (PCA) is used to show the strong correlation characteristics among the product quality attributes including aggregates, fragments, charge variants, and glycans. Then, partial least square regression (PLS1 and PLS2) is applied to predict the product quality variables based on process information (one by one or simultaneously). The comparison of those two modeling techniques shows that a single (PLS2) model is capable of revealing the interrelationship of the process characteristics to the large set product quality variables. In order to show the dynamic evolution of the process predictability separate models are defined at different time points showing that several product quality attributes are mainly driven by the media composition and, hence, can be decently predicted from early on in the process, while others are strongly affected by process parameter changes during the process. Finally, by coupling the PLS2 models with a genetic algorithm first the model performance can be further improved and, most importantly, the interpretation of the large-dimensioned process-product-interrelationship can be significantly simplified. The generally applicable toolset presented in this case study provides a solid basis for decision making and process optimization throughout process development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1368-1380, 2017. © 2017 American Institute of Chemical Engineers.

  6. Conglutinin exhibits a complement-dependent enhancement of the respiratory burst of phagocytes stimulated by E. coli

    DEFF Research Database (Denmark)

    Friis, P; Svehag, S E; Andersen, Ove

    1991-01-01

    . Conglutinin enhances, in a dose-dependent manner, the respiratory burst of spleen cells stimulated with serum-opsonized Escherichia coli. The enhancement was only demonstrable in the presence of a functional complement system. The conglutinin-mediated enhancement of the respiratory burst was inhibited......Conglutinin is a mammalian C-type lectin which shows anti-bacterial activity when tested in vivo and in vitro. This study concerns the effect of conglutinin on the respiratory burst of murine spleen cells, using a chemiluminescence assay for measurement of generated reactive oxygen metabolites...

  7. A combination of p53-activating APR-246 and phosphatidylserine-targeting antibody potently inhibits tumor development in hormone-dependent mutant p53-expressing breast cancer xenografts

    Directory of Open Access Journals (Sweden)

    Liang Y

    2018-03-01

    Full Text Available Yayun Liang,1 Benford Mafuvadze,1 Cynthia Besch-Williford,2 Salman M Hyder1 1Deparment of Biomedical Sciences and Dalton Cardiovascular Research Center, Columbia, MO, USA; 2IDEXX BioResearch, Columbia, MO, USA Background: Between 30 and 40% of human breast cancers express a defective tumor suppressor p53 gene. Wild-type p53 tumor suppressor protein promotes cell-cycle arrest and apoptosis and inhibits vascular endothelial growth factor–dependent angiogenesis, whereas mutant p53 protein (mtp53 lacks these functions, resulting in tumor cell survival and metastasis. Restoration of p53 function is therefore a promising drug-targeted strategy for combating mtp53-expressing breast cancer. Methods: In this study, we sought to determine whether administration of APR-246, a small-molecule drug that restores p53 function, in combination with 2aG4, an antibody that targets phosphatidylserine residues on tumor blood vessels and disrupts tumor vasculature, effectively inhibits advanced hormone-dependent breast cancer tumor growth. Results: APR-246 reduced cell viability in mtp53-expressing BT-474 and T47-D human breast cancer cells in vitro, and significantly induced apoptosis in a dose-dependent manner. However, APR-246 did not reduce cell viability in MCF-7 breast cancer cells, which express wild-type p53. We next examined APR-246’s anti-tumor effects in vivo using BT-474 and T47-D tumor xenografts established in female nude mice. Tumor-bearing mice were treated with APR-246 and/or 2aG4 and tumor volume followed over time. Tumor growth was more effectively suppressed by combination treatment than by either agent alone, and combination therapy completely eradicated some tumors. Immunohistochemistry analysis of tumor tissue sections demonstrated that combination therapy more effectively induced apoptosis and reduced cell proliferation in tumor xenografts than either agent alone. Importantly, combination therapy dramatically reduced the density of blood

  8. Novel monoclonal antibodies to study tissue regeneration in planarians.

    Science.gov (United States)

    Ross, Kelly G; Omuro, Kerilyn C; Taylor, Matthew R; Munday, Roma K; Hubert, Amy; King, Ryan S; Zayas, Ricardo M

    2015-01-21

    Planarians are an attractive model organism for studying stem cell-based regeneration due to their ability to replace all of their tissues from a population of adult stem cells. The molecular toolkit for planarian studies currently includes the ability to study gene function using RNA interference (RNAi) and observe gene expression via in situ hybridizations. However, there are few antibodies available to visualize protein expression, which would greatly enhance analysis of RNAi experiments as well as allow further characterization of planarian cell populations using immunocytochemistry and other immunological techniques. Thus, additional, easy-to-use, and widely available monoclonal antibodies would be advantageous to study regeneration in planarians. We have created seven monoclonal antibodies by inoculating mice with formaldehyde-fixed cells isolated from dissociated 3-day regeneration blastemas. These monoclonal antibodies can be used to label muscle fibers, axonal projections in the central and peripheral nervous systems, two populations of intestinal cells, ciliated cells, a subset of neoblast progeny, and discrete cells within the central nervous system as well as the regeneration blastema. We have tested these antibodies using eight variations of a formaldehyde-based fixation protocol and determined reliable protocols for immunolabeling whole planarians with each antibody. We found that labeling efficiency for each antibody varies greatly depending on the addition or removal of tissue processing steps that are used for in situ hybridization or immunolabeling techniques. Our experiments show that a subset of the antibodies can be used alongside markers commonly used in planarian research, including anti-SYNAPSIN and anti-SMEDWI, or following whole-mount in situ hybridization experiments. The monoclonal antibodies described in this paper will be a valuable resource for planarian research. These antibodies have the potential to be used to better understand

  9. Enhancement of antitumor activity by using a fully human gene encoding a single-chain fragmented antibody specific for carcinoembryonic antigen

    Science.gov (United States)

    Kuroki, Motomu; Kuroki, Masahide

    2017-01-01

    Human leukocyte antigen and/or costimulatory molecules are frequently lacking in metastatic tumor cells, and thus tumor cells are able to escape from the immune system. Although lymphocytes with a chimeric antigen receptor (CAR) is a promising approach for overcoming this challenge in cancer immunotherapy, administration of modified T cells alone often demonstrates little efficacy in patients. Therefore, in order to enhance the antitumor activity of immune cells in the cancer microenvironment, we used lymphocytes expressing CAR in combination with a fusion protein of IL-2 that contained the single-chain fragmented antibody (scFv) specific for the carcinoembryonic antigen. Among a series of CAR constructs, with or without a spacer and the intracellular domain of CD28, the CAR construct containing CD8α, CD28, and CD3ζ most effectively activated and expressed INF-γ in CAR-bearing T cells. Furthermore, in comparison with free IL-2, the combination of peripheral blood mononuclear cells expressing CAR and the fusion protein containing IL-2 significantly enhanced the antitumor activity against MKN-45 cells, a human gastric cancer cell line. In conclusion, this novel combination therapy of CAR and a fusion protein consisting of a functional cytokine and a fully human scFv may be a promising approach for adoptive cancer immunotherapy. PMID:28860806

  10. Monoclonal Antibodies Specific for the V2, V3, CD4-Binding Site, and gp41 of HIV-1 Mediate Phagocytosis in a Dose-Dependent Manner.

    Science.gov (United States)

    Musich, Thomas; Li, Liuzhe; Liu, Lily; Zolla-Pazner, Susan; Robert-Guroff, Marjorie; Gorny, Miroslaw K

    2017-04-15

    In light of the weak or absent neutralizing activity mediated by anti-V2 monoclonal antibodies (MAbs), we tested whether they can mediate Ab-dependent cellular phagocytosis (ADCP), which is an important element of anti-HIV-1 immunity. We tested six anti-V2 MAbs and compared them with 21 MAbs specific for V3, the CD4-binding site (CD4bs), and gp41 derived from chronically HIV-1-infected individuals and produced by hybridoma cells. ADCP activity was measured by flow cytometry using uptake by THP-1 monocytic cells of fluorescent beads coated with gp120, gp41, BG505 SOSIP.664, or BG505 DS-SOSIP.664 complexed with MAbs. The measurement of ADCP activity by the area under the curve showed significantly higher activity of anti-gp41 MAbs than of the members of the three other groups of MAbs tested using beads coated with monomeric gp41 or gp120; anti-V2 MAbs were dominant compared to anti-V3 and anti-CD4bs MAbs against clade C gp120 ZM109 ADCP activity mediated by V2 and V3 MAbs was positive against stabilized DS-SOSIP.664 trimer but negligible against SOSIP.664 targets, suggesting that a closed envelope conformation better exposes the variable loops. Two IgG3 MAbs against the V2 and V3 regions displayed dominant ADCP activity compared to a panel of IgG1 MAbs. This superior ADCP activity was confirmed when two of three recombinant IgG3 anti-V2 MAbs were compared to their IgG1 counterparts. The study demonstrated dominant ADCP activity of anti-gp41 against monomers but not trimers, with some higher activity of anti-V2 MAbs than of anti-V3 and anti-CD4bs MAbs. The ability to mediate ADCP suggests a mechanism by which anti-HIV-1 envelope Abs can contribute to protective efficacy. IMPORTANCE Anti-V2 antibodies (Abs) correlated with reduced risk of HIV-1 infection in recipients of the RV144 vaccine, suggesting that they play a protective role, but a mechanism providing such protection remains to be determined. The rare and weak neutralizing activities of anti-V2 MAbs prompted us

  11. Dengue Virus Nonstructural Protein 1-Induced Antibodies Cross-React with Human Plasminogen and Enhance Its Activation.

    Science.gov (United States)

    Chuang, Yung-Chun; Lin, Jessica; Lin, Yee-Shin; Wang, Shuying; Yeh, Trai-Ming

    2016-02-01

    Dengue virus (DENV) infection is the most common mosquito-borne viral disease, and it can cause life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Abnormal activation of the coagulation and fibrinolysis system is one of the hallmarks of DHF/DSS. However, the mechanism underlying hemorrhage in DHF/DSS remains elusive. In previous studies, plasminogen (Plg) cross-reactive Abs, which can recognize DENV nonstructural protein (NS) 1, have been found in dengue patients. However, it is unclear whether these Abs are indeed induced by DENV NS1. Thus, we immunized mice with recombinant NS1 from both bacteria and drosophila to determine whether NS1 can induce Plg cross-reactive Abs. The results from the NS1-immunized mouse sera indicated that NS1 immunization induced Abs that could cross-react with Plg. To study the effects of these NS1-induced Plg cross-reactive Abs on fibrinolysis, we isolated several Plg cross-reactive anti-NS1 mAbs from these mice and found that some of them could enhance Plg activation. In addition, epitope mapping with a phage-displayed random peptide library revealed that one of these mAbs (2A5) could recognize NS1 C-terminal residues 305-311, which share sequence homology with Plg residues 590-597. A synthetic peptide of NS1 residues 305-311 could inhibit the binding of both 2A5 and its Fab to Plg and its enhanced activation. Thus, our results suggest that DENV NS1 can induce Plg cross-reactive Abs through molecular mimicry, which can enhance Plg activation and may contribute to the pathogenesis of DHF/DSS. Copyright © 2016 by The American Association of Immunologists, Inc.

  12. The Human Antibody Fragment DIATHIS1 Specific for CEACAM1 Enhances Natural Killer Cell Cytotoxicity Against Melanoma Cell Lines In Vitro

    Science.gov (United States)

    Dupuis, Maria L.; Soriani, Alessandra; Ricci, Biancamaria; Dominici, Sabrina; Moricoli, Diego; Ascione, Alessandro; Santoni, Angela; Magnani, Mauro; Cianfriglia, Maurizio

    2015-01-01

    Several lines of evidence show that de novo expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is strongly associated with reduced disease-free survival of patients affected by metastatic melanoma. Previously published investigations report that homophilic interactions between CEACAM1 expressed on natural killer (NK) cells and tumors inhibit the NK cell-mediated killing independently of major histocompatibility complex class I recognition. This biological property can be physiologically relevant in metastatic melanoma because of the increased CEACAM1 expression observed on NK cells from some patients. Moreover, this inhibitory mechanism in many cases might hinder the efficacy of immunotherapeutic treatments of CEACAM1+ malignancies because of tumor evasion by activated effector cells. In the present study, we designed an in vitro experimental model showing that the human single-chain variable fragment (scFv) DIATHIS1 specific for CEACAM1 is able to enhance the lytic machinery of NK cells against CEACAM1+ melanoma cells. The coincubation of the scFv DIATHIS1 with CEACAM1+ melanoma cells and NK-92 cell line significantly increases the cell-mediated cytotoxicity. Moreover, pretreatment of melanoma cells with scFv DIATHIS1 promotes the activation and the degranulation capacity of in vitro–expanded NK cells from healthy donors. It is interesting to note that the melanoma cell line MelC and the primary melanoma cells STA that respond better to DIATHIS1 treatment, express higher relative levels of CEACAM1-3L and CEACAM1-3S splice variants isoforms compared with Mel501 cells that are less responsive to DIATHIS1-induced NK cell–mediated cytotoxicity. Taken together, our results suggest that the fully human antibody fragment DIATHIS1 originated by biopanning approach from a phage antibody library may represent a relevant biotechnological platform to design and develop completely human antimelanoma therapeutics of biological origin. PMID

  13. Antibodies to biotinylated red blood cells in adults and infants: improved detection, partial characterization, and dependence on red blood cell-biotin dose.

    Science.gov (United States)

    Schmidt, Robert L; Mock, Donald M; Franco, Robert S; Cohen, Robert M; North, Anne K; Cancelas, José A; Geisen, Christof; Strauss, Ronald G; Vlaar, Alexander P; Nalbant, Demet; Widness, John A

    2017-06-01

    Biotin-labeled red blood cells (BioRBCs) are used for in vivo kinetic studies. Because BioRBC dosing occasionally induces antibodies, a sensitive and specific anti-BioRBC detection assay is needed. Aims were to 1) develop a gel card assay to evaluate existing, naturally occurring and BioRBC-induced plasma antibodies, 2) compare gel card and tube agglutination detection results, and 3) test for a relationship of antibody induction and BioRBC dose. Reagent BioRBCs were prepared using sulfo-NHS biotin ranging from densities 18 (BioRBC-18) to 1458 (BioRBC-1458) µg/mL RBCs. Among BioRBC-exposed subjects, gel card and tube agglutination results were concordant in 21 of 22 adults and all 19 infant plasma samples. Gel card antibody detection sensitivity was more than 10-fold greater than tube agglutination. Twelve to 16 weeks after BioRBC exposure, induced anti-antibodies were detected by gel card in three of 26 adults (12%) at reagent densities BioRBC-256 or less, but in none of 41 infants. Importantly, induced anti-BioRBC antibodies were associated with higher BioRBC dose (p = 0.008); no antibodies were detected in 18 subjects who received BioRBC doses less than or equal to BioRBC-18. For noninduced BioRBC antibodies, six of 1125 naïve adults (0.3%) and none of 46 naïve infants demonstrated existing anti-BioRBC antibodies using reagent BioRBC-140 or -162. Existing anti-BioRBCs were all neutralized by biotin compounds, while induced antibodies were not. The gel card assay is more sensitive than the tube agglutination assay. We recommend reagent BioRBC-256 for identifying anti-BioRBCs. Use of a low total RBC biotin label dose (≤ BioRBC-18) may minimize antibody induction. © 2017 AABB.

  14. EpCAM antibody-conjugated mesoporous silica nanoparticles to enhance the anticancer efficacy of carboplatin in retinoblastoma.

    Science.gov (United States)

    Qu, Wei; Meng, Bo; Yu, Yangyang; Wang, Shaowei

    2017-07-01

    Retinoblastoma (RB) is a rare type of tumor which occurs in the intraocular spaces of eye. Carboplatin (CRB) has been used for the treatment of RB along with focal therapy. In this study, we have demonstrated the preparation of EpCAM-conjugated mesoporous silica nanoparticles for the successful delivery of carboplatin (CRB) to the retinoblastoma cells. The particles size and morphology was nanosized and spherical and exhibited a controlled release kinetics. Cellular uptake studies reveal that enhanced internalization was observed for EpCMSN compared to that of CMSN. The remarkable difference in cellular uptake might be attributed to the specific receptor mediated cellular internalization for EpCMSN compared to that of CMSN which might entered the cells by passive diffusion. Consistently, EpCMSN exhibited a superior anticancer effect in retinoblastoma cells. The IC50 value of EpCMSN was 1.38μg/ml which is significantly lesser compared to that of free CRB (3.26μg/ml). EpCMSN resulted in 2-fold increase in caspase-3 level compared to that of free CRB. The higher targeting effect of EpCAM receptor resulted in enhanced apoptosis of cancer cells after treatment with EpCMSN in retinoblastoma cells. Taken together, EpCAM conjugated MSN might be advantageous for the targeted treatment of receptor-overexpressed ocular malignancies like retinoblastoma. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Enhancement of striatum-dependent memory by conditioned fear is mediated by beta-adrenergic receptors in the basolateral amygdala

    Directory of Open Access Journals (Sweden)

    Travis D. Goode

    2016-06-01

    Full Text Available Emotional arousal can have a profound impact on various learning and memory processes. For example, unconditioned emotional stimuli (e.g., predator odor or anxiogenic drugs enhance dorsolateral striatum (DLS-dependent habit memory. These effects critically depend on a modulatory role of the basolateral complex of the amygdala (BLA. Recent work indicates that, like unconditioned emotional stimuli, exposure to an aversive conditioned stimulus (CS (i.e., a tone previously paired with shock can also enhance consolidation of DLS-dependent habit memory. The present experiments examined whether noradrenergic activity, particularly within the BLA, is required for a fear CS to enhance habit memory consolidation. First, rats underwent a fear conditioning procedure in which a tone CS was paired with an aversive unconditioned stimulus. Over the course of the next five days, rats received training in a DLS-dependent water plus-maze task, in which rats were reinforced to make a consistent body-turn response to reach a hidden escape platform. Immediately after training on days 1–3, rats received post-training systemic (Experiment 1 or intra-BLA (Experiment 2 administration of the β-adrenoreceptor antagonist, propranolol. Immediately after drug administration, half of the rats were re-exposed to the tone CS in the conditioning context (without shock. Post-training CS exposure enhanced consolidation of habit memory in vehicle-treated rats, and this effect was blocked by peripheral (Experiment 1 or intra-BLA (Experiment 2 propranolol administration. The present findings reveal that noradrenergic activity within the BLA is critical for the enhancement of DLS-dependent habit memory as a result of exposure to conditioned emotional stimuli.

  16. Monoclonal antibody "gold rush".

    Science.gov (United States)

    Maggon, Krishan

    2007-01-01

    The market, sales and regulatory approval of new human medicines, during the past few years, indicates increasing number and share of new biologics and emergence of new multibillion dollar molecules. The global sale of monoclonal antibodies in 2006 were $20.6 billion. Remicade had annual sales gain of $1 billion during the past 3 years and five brands had similar increase in 2006. Rituxan with 2006 sales of $4.7 billion was the best selling monoclonal antibody and biological product and the 6th among the top selling medicinal brand. It may be the first biologic and monoclonal antibody to reach $10 billion annual sales in the near future. The strong demand from cancer and arthritis patients has surpassed almost all commercial market research reports and sales forecast. Seven monoclonal antibody brands in 2006 had sales exceeding $1 billion. Humanized or fully human monoclonal antibodies with low immunogenicity, enhanced antigen binding and reduced cellular toxicity provide better clinical efficacy. The higher technical and clinical success rate, overcoming of technical hurdles in large scale manufacturing, low cost of market entry and IND filing, use of fully human and humanized monoclonal antibodies has attracted funds and resources towards R&D. Review of industry research pipeline and sales data during the past 3 years indicate a real paradigm shift in industrial R&D from pharmaceutical to biologics and monoclonal antibodies. The antibody bandwagon has been joined by 200 companies with hundreds of new projects and targets and has attracted billions of dollars in R&D investment, acquisitions and licensing deals leading to the current Monoclonal Antibody Gold Rush.

  17. Antiprothrombin Antibodies

    Directory of Open Access Journals (Sweden)

    Polona Žigon

    2015-05-01

    Full Text Available In patients with the antiphospholipid syndrome (APS, the presence of a group of pathogenic autoantibodies called antiphospholipid antibodies causes thrombosis and pregnancy complications. The most frequent antigenic target of antiphospholipid antibodies are phospholipid bound β2-glycoprotein 1 (β2GPI and prothrombin. The international classification criteria for APS connect the occurrence of thrombosis and/or obstetric complications together with the persistence of lupus anticoagulant, anti-cardiolipin antibodies (aCL and antibodies against β2GPI (anti-β2GPI into APS. Current trends for the diagnostic evaluation of APS patients propose determination of multiple antiphospholipid antibodies, among them also anti-prothrombin antibodies, to gain a common score which estimates the risk for thrombosis in APS patients. Antiprothrombin antibodies are common in APS patients and are sometimes the only antiphospholipid antibodies being elevated. Methods for their determination differ and have not yet been standardized. Many novel studies confirmed method using phosphatidylserine/prothrombin (aPS/PT ELISA as an antigen on solid phase encompass higher diagnostic accuracy compared to method using prothrombin alone (aPT ELISA. Our research group developed an in-house aPS/PT ELISA with increased analytical sensitivity which enables the determination of all clinically relevant antiprothrombin antibodies. aPS/PT exhibited the highest percentage of lupus anticoagulant activity compared to aCL and anti-β2GPI. aPS/PT antibodies measured with the in-house method associated with venous thrombosis and presented the strongest independent risk factor for the presence of obstetric complications among all tested antiphospholipid antibodies

  18. Monoclonal Antibodies, Derived from Humans Vaccinated with the RV144 HIV Vaccine Containing the HVEM Binding Domain of Herpes Simplex Virus (HSV) Glycoprotein D, Neutralize HSV Infection, Mediate Antibody-Dependent Cellular Cytotoxicity, and Protect Mice from Ocular Challenge with HSV-1.

    Science.gov (United States)

    Wang, Kening; Tomaras, Georgia D; Jegaskanda, Sinthujan; Moody, M Anthony; Liao, Hua-Xin; Goodman, Kyle N; Berman, Phillip W; Rerks-Ngarm, Supachai; Pitisuttithum, Punnee; Nitayapan, Sorachai; Kaewkungwal, Jaranit; Haynes, Barton F; Cohen, Jeffrey I

    2017-10-01

    The RV144 HIV vaccine trial included a recombinant HIV glycoprotein 120 (gp120) construct fused to a small portion of herpes simplex virus 1 (HSV-1) glycoprotein D (gD) so that the first 40 amino acids of gp120 were replaced by the signal sequence and the first 27 amino acids of the mature form of gD. This region of gD contains most of the binding site for HVEM, an HSV receptor important for virus infection of epithelial cells and lymphocytes. RV144 induced antibodies to HIV that were partially protective against infection, as well as antibodies to HSV. We derived monoclonal antibodies (MAbs) from peripheral blood B cells of recipients of the RV144 HIV vaccine and showed that these antibodies neutralized HSV-1 infection in cells expressing HVEM, but not the other major virus receptor, nectin-1. The MAbs mediated antibody-dependent cellular cytotoxicity (ADCC), and mice that received the MAbs and were then challenged by corneal inoculation with HSV-1 had reduced eye disease, shedding, and latent infection. To our knowledge, this is the first description of MAbs derived from human recipients of a vaccine that specifically target the HVEM binding site of gD. In summary, we found that monoclonal antibodies derived from humans vaccinated with the HVEM binding domain of HSV-1 gD (i) neutralized HSV-1 infection in a cell receptor-specific manner, (ii) mediated ADCC, and (iii) reduced ocular disease in virus-infected mice. IMPORTANCE Herpes simplex virus 1 (HSV-1) causes cold sores and neonatal herpes and is a leading cause of blindness. Despite many trials, no HSV vaccine has been approved. Nectin-1 and HVEM are the two major cellular receptors for HSV. These receptors are expressed at different levels in various tissues, and the role of each receptor in HSV pathogenesis is not well understood. We derived human monoclonal antibodies from persons who received the HIV RV144 vaccine that contained the HVEM binding domain of HSV-1 gD fused to HIV gp120. These antibodies were

  19. SU-E-T-44: Angular Dependence of Surface Dose Enhancement Measured On Several Inhomogeneities Using Radiochromic EBT3 Films

    Energy Technology Data Exchange (ETDEWEB)

    Jansen, A; Schoenfeld, A; Poppinga, D; Chofor, N; Poppe, B [University of Oldenburg, Oldenburg (Germany); Pius Hospital Oldenburg, Oldenburg (Germany)

    2014-06-01

    Purpose: The quantification of the relative surface dose enhancement in dependence on the angle of incidence and the atomic number Z of the surface material. Methods: Experiments were performed with slabs made of aluminum, titanium, copper, silver, dental gold and lead. The metal slabs with equal sizes of 1.0×8.0×8.8mm{sup 3} were embedded in an Octavius 4D phantom (PTW Freiburg, Germany). Radiochromic EBT3 films were used to measure the surface dose for angles of incidence ranging from 0° to 90°. The setup with the metals slabs at the isocenter was irradiated with acceleration voltages of 6MV and 10MV. Water reference measurements were taken under equal conditions. Results: The surface dose enhancement is highest for angles of incidence below 30° and drops significantly for higher. The surface dose enhancement produced by lead and dental gold at 6MV showed a peak of 65%. At 90°, the surface dose enhancement dropped to 15% for both materials. The surface dose enhancements for silver, copper, titanium and aluminum were 45%, 32%, 22% and 12% at 0°, respectively. At an angle of incidence of 80°, the values dropped to 22%, 18%, 12% und 6%. The values for 10MV were very similar. Lead and dental gold showed peaks of 65% und 60%. Their values dropped to 18% at an angle of 90°. The surface dose enhancements for silver, copper, titanium and aluminum were 45%, 30%, 20% and 8% at 0°. At 80° the values dropped to 30%, 20%, 12% and 5%. A dependence of the magnitude of the surface dose enhancement on the atomic number of the surface material can be seen, which is in consistence with literature. Conclusion: The results show that the surface dose enhancements near implant materials with high Z-values should be taken into consideration in radio therapy, even when the angle of incidence is flat.

  20. SU-E-T-44: Angular Dependence of Surface Dose Enhancement Measured On Several Inhomogeneities Using Radiochromic EBT3 Films

    International Nuclear Information System (INIS)

    Jansen, A; Schoenfeld, A; Poppinga, D; Chofor, N; Poppe, B

    2014-01-01

    Purpose: The quantification of the relative surface dose enhancement in dependence on the angle of incidence and the atomic number Z of the surface material. Methods: Experiments were performed with slabs made of aluminum, titanium, copper, silver, dental gold and lead. The metal slabs with equal sizes of 1.0×8.0×8.8mm 3 were embedded in an Octavius 4D phantom (PTW Freiburg, Germany). Radiochromic EBT3 films were used to measure the surface dose for angles of incidence ranging from 0° to 90°. The setup with the metals slabs at the isocenter was irradiated with acceleration voltages of 6MV and 10MV. Water reference measurements were taken under equal conditions. Results: The surface dose enhancement is highest for angles of incidence below 30° and drops significantly for higher. The surface dose enhancement produced by lead and dental gold at 6MV showed a peak of 65%. At 90°, the surface dose enhancement dropped to 15% for both materials. The surface dose enhancements for silver, copper, titanium and aluminum were 45%, 32%, 22% and 12% at 0°, respectively. At an angle of incidence of 80°, the values dropped to 22%, 18%, 12% und 6%. The values for 10MV were very similar. Lead and dental gold showed peaks of 65% und 60%. Their values dropped to 18% at an angle of 90°. The surface dose enhancements for silver, copper, titanium and aluminum were 45%, 30%, 20% and 8% at 0°. At 80° the values dropped to 30%, 20%, 12% and 5%. A dependence of the magnitude of the surface dose enhancement on the atomic number of the surface material can be seen, which is in consistence with literature. Conclusion: The results show that the surface dose enhancements near implant materials with high Z-values should be taken into consideration in radio therapy, even when the angle of incidence is flat

  1. Enhanced and persistent antibody response against homologous and heterologous strains elicited by a MF59-adjuvanted influenza vaccine in infants and young children.

    Science.gov (United States)

    Nolan, Terry; Bravo, Lulu; Ceballos, Ana; Mitha, Essack; Gray, Glenda; Quiambao, Beatriz; Patel, Sanjay S; Bizjajeva, Svetlana; Bock, Hans; Nazaire-Bermal, Nancy; Forleo-Neto, Eduardo; Cioppa, Giovanni Della; Narasimhan, Vas

    2014-10-21

    Non-adjuvanted seasonal influenza vaccines show only modest efficacy in young children. This study compared the immunogenicity, reactogenicity and safety of the MF59-adjuvanted trivalent subunit vaccine (aTIV) with two non-adjuvanted trivalent vaccines, TIV-1, the non-adjuvanted version of aTIV, and TIV-2, a split virion vaccine. 6078 children received two doses of aTIV (n=3125), TIV-1 (n=1479), or TIV-2 (n=1474) four weeks apart (Days 1 and 29). Children aged 6 to vaccination (Day 50), the aTIV group showed significantly higher geometric mean HI titers and seroconversion rates than the TIV-1 or TIV-2 groups against all homologous and heterologous strains. The difference was enhanced at HI titers ≥110. aTIV elicited a faster, more persistent antibody response, with significantly higher titers in the aTIV group after one vaccination (Day 29) and after six months (Day 209) than in either TIV group. aTIV was more reactogenic than were TIV-1 and TIV-2 but rates of severe adverse events were very low for all three vaccines. In infants and young children, the MF59-adjuvanted vaccine induced substantially faster (after one dose), higher, persistent HI titers than the non-adjuvanted vaccines, with consistently higher seroprotection rates at increased threshold HI titers. This trial is registered at clinicaltrials.gov: NCT01346592. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Localization of CD26/DPPIV in nucleus and its nuclear translocation enhanced by anti-CD26 monoclonal antibody with anti-tumor effect

    Directory of Open Access Journals (Sweden)

    Sakamoto Michiie

    2009-06-01

    Full Text Available Abstract Background CD26 is a type II, cell surface glycoprotein known as dipeptidyl peptidase (DPP IV. Previous studies have revealed CD26 expression in T cell leukemia/lymphoma and malignant mesothelioma, and an inhibitory effect of anti-CD26 monoclonal antibody (mAb against the growth of CD26+ cancer cells in vitro and in vivo. The function of CD26 in tumor development is unknown and the machinery with which the CD26 mAb induces its anti-tumor effect remains uncharacterized. Results The localization of CD26 in the nucleus of T cell leukemia/lymphoma cells and mesothelioma cells was shown by biochemical and immuno-electron microscopic analysis. The DPPIV enzyme activity was revealed in the nuclear fraction of T cell leukemia/lymphoma cells. These expressions of intra-nuclear CD26 were augmented by treatment with the CD26 mAb, 1F7, with anti-tumor effect against the CD26+ T cell leukemia/lymphoma cells. In contrast, the CD26 mAb, 5F8, without anti-tumor effect, did not augment CD26 expressions in the nucleus. Biotin-labeled, cell surface CD26 translocated into the nucleus constantly, and this translocation was enhanced with 1F7 treatment but not with 5F8. Conclusion These results indicate that the intra-nuclear CD26 which moves from plasma membrane may play certain roles in cell growth of human cancer cells.

  3. Enhancement of antitumor activity by using a fully human gene encoding a single-chain fragmented antibody specific for carcinoembryonic antigen

    Directory of Open Access Journals (Sweden)

    Shibaguchi H

    2017-08-01

    Full Text Available Hirotomo Shibaguchi,1,* Naixiang Luo,1,* Naoto Shirasu,1,* Motomu Kuroki,2 Masahide Kuroki1 1Department of Biochemistry, Faculty of Medicine, Fukuoka University, Fukuoka, Japan; 2School of Nursing, Faculty of Medicine, Fukuoka University, Fukuoka, Japan *These authors equally contributed to this work Abstract: Human leukocyte antigen and/or costimulatory molecules are frequently lacking in metastatic tumor cells, and thus tumor cells are able to escape from the immune system. Although lymphocytes with a chimeric antigen receptor (CAR is a promising approach for overcoming this challenge in cancer immunotherapy, administration of modified T cells alone often demonstrates little efficacy in patients. Therefore, in order to enhance the antitumor activity of immune cells in the cancer microenvironment, we used lymphocytes expressing CAR in combination with a fusion protein of IL-2 that contained the single-chain fragmented antibody (scFv specific for the carcinoembryonic antigen. Among a series of CAR constructs, with or without a spacer and the intracellular domain of CD28, the CAR construct containing CD8α, CD28, and CD3ζ most effectively activated and expressed INF-γ in CAR-bearing T cells. Furthermore, in comparison with free IL-2, the combination of peripheral blood mononuclear cells expressing CAR and the fusion protein containing IL-2 significantly enhanced the antitumor activity against MKN-45 cells, a human gastric cancer cell line. In conclusion, this novel combination therapy of CAR and a fusion protein consisting of a functional cytokine and a fully human scFv may be a promising approach for adoptive cancer immunotherapy. Keywords: chimeric antigen receptor, fusion protein, human scFv, CEA, combination therapy

  4. Antibody Production with Synthetic Peptides.

    Science.gov (United States)

    Lee, Bao-Shiang; Huang, Jin-Sheng; Jayathilaka, Lasanthi P; Lee, Jenny; Gupta, Shalini

    2016-01-01

    Peptides (usually 10-20 amino acid residues in length) can be used as effectively as proteins in raising antibodies producing both polyclonal and monoclonal antibodies routinely with titers higher than 20,000. Peptide antigens do not function as immunogens unless they are conjugated to proteins. Production of high quality antipeptide antibodies is dependent upon peptide sequence selection, the success of peptide synthesis, peptide-carrier protein conjugation, the humoral immune response in the host animal, the adjuvant used, the peptide dose administered, the injection method, and the purification of the antibody. Peptide sequence selection is probably the most critical step in the production of antipeptide antibodies. Although the process for designing peptide antigens is not exact, several guidelines and computational B-cell epitope prediction methods can help maximize the likelihood of producing antipeptide antibodies that recognize the protein. Antibodies raised by peptides have become essential tools in life science research. Virtually all phospho-specific antibodies are now produced using phosphopeptides as antigens. Typically, 5-20 mg of peptide is enough for antipeptide antibody production. It takes 3 months to produce a polyclonal antipeptide antibody in rabbits that yields ~100 mL of serum which corresponds to ~8-10 mg of the specific antibody after affinity purification using a peptide column.

  5. On-Line Dependability Enhancement of Multiprocessor SoCs by Resource Management

    NARCIS (Netherlands)

    ter Braak, T.D.; Burgess, S.T.; Hurskainen, H.; Kerkhoff, Hans G.; Vermeulen, B.; Zhang, X.

    2010-01-01

    This paper describes a new approach towards dependable design of homogeneous multi-processor SoCs in an example satellite-navigation application. First, the NoC dependability is functionally verified via embedded software. Then the Xentium processor tiles are periodically verified via on-line

  6. Nerve growth factor enhances the CRE-dependent transcriptional activity activated by nobiletin in PC12 cells.

    Science.gov (United States)

    Takito, Jiro; Kimura, Junko; Kajima, Koji; Uozumi, Nobuyuki; Watanabe, Makoto; Yokosuka, Akihito; Mimaki, Yoshihiro; Nakamura, Masanori; Ohizumi, Yasushi

    2016-07-01

    Prevention and treatment of Alzheimer disease are urgent problems for elderly people in developed countries. We previously reported that nobiletin, a poly-methoxylated flavone from the citrus peel, improved the symptoms in various types of animal models of memory loss and activated the cAMP responsive element (CRE)-dependent transcription in PC12 cells. Nobiletin activated the cAMP/PKA/MEK/Erk/MAPK signaling pathway without using the TrkA signaling activated by nerve growth factor (NGF). Here, we examined the effect of combination of nobiletin and NGF on the CRE-dependent transcription in PC12 cells. Although NGF alone had little effect on the CRE-dependent transcription, NGF markedly enhanced the CRE-dependent transcription induced by nobiletin. The NGF-induced enhancement was neutralized by a TrkA antagonist, K252a. This effect of NGF was effective on the early signaling event elicited by nobiletin. These results suggested that there was crosstalk between NGF and nobiletin signaling in activating the CRE-dependent transcription in PC12 cells.

  7. Ouabain enhancement of compound 48/80 induced histamine secretion from rat peritoneal mast cells: dependence on extracellular sodium

    DEFF Research Database (Denmark)

    Knudsen, T; Bertelsen, Niels Haldor; Johansen, Torben

    1992-01-01

    a measure of the Na(+)-K+ pump activity of the cells. Ouabain caused an immediate inhibition of the pump activity and a time-dependent increase in histamine secretion in the absence of extracellular calcium. No effect on the secretion was observed in the presence of calcium. The effect of ouabain......-free medium, the pump activity was inhibited and the enhancement by ouabain of the secretion of histamine was blocked. A less marked inhibition of the pump was found in a calcium-free medium containing magnesium. The inhibition exerted by magnesium was concentration-dependent (0-5 mM) as was the counteraction...

  8. Concerted activity of IgG1 antibodies and IL-4/IL-25-dependent effector cells trap helminth larvae in the tissues following vaccination with defined secreted antigens, providing sterile immunity to challenge infection.

    Directory of Open Access Journals (Sweden)

    James P Hewitson

    2015-03-01

    Full Text Available Over 25% of the world's population are infected with helminth parasites, the majority of which colonise the gastrointestinal tract. However, no vaccine is yet available for human use, and mechanisms of protective immunity remain unclear. In the mouse model of Heligmosomoides polygyrus infection, vaccination with excretory-secretory (HES antigens from adult parasites elicits sterilising immunity. Notably, three purified HES antigens (VAL-1, -2 and -3 are sufficient for effective vaccination. Protection is fully dependent upon specific IgG1 antibodies, but passive transfer confers only partial immunity to infection, indicating that cellular components are also required. Moreover, immune mice show greater cellular infiltration associated with trapping of larvae in the gut wall prior to their maturation. Intra-vital imaging of infected intestinal tissue revealed a four-fold increase in extravasation by LysM+GFP+ myeloid cells in vaccinated mice, and the massing of these cells around immature larvae. Mice deficient in FcRγ chain or C3 complement component remain fully immune, suggesting that in the presence of antibodies that directly neutralise parasite molecules, the myeloid compartment may attack larvae more quickly and effectively. Immunity to challenge infection was compromised in IL-4Rα- and IL-25-deficient mice, despite levels of specific antibody comparable to immune wild-type controls, while deficiencies in basophils, eosinophils or mast cells or CCR2-dependent inflammatory monocytes did not diminish immunity. Finally, we identify a suite of previously uncharacterised heat-labile vaccine antigens with homologs in human and veterinary parasites that together promote full immunity. Taken together, these data indicate that vaccine-induced immunity to intestinal helminths involves IgG1 antibodies directed against secreted proteins acting in concert with IL-25-dependent Type 2 myeloid effector populations.

  9. Concerted Activity of IgG1 Antibodies and IL-4/IL-25-Dependent Effector Cells Trap Helminth Larvae in the Tissues following Vaccination with Defined Secreted Antigens, Providing Sterile Immunity to Challenge Infection

    Science.gov (United States)

    Hewitson, James P.; Filbey, Kara J.; Esser-von Bieren, Julia; Camberis, Mali; Schwartz, Christian; Murray, Janice; Reynolds, Lisa A.; Blair, Natalie; Robertson, Elaine; Harcus, Yvonne; Boon, Louis; Huang, Stanley Ching-Cheng; Yang, Lihua; Tu, Yizheng; Miller, Mark J.; Voehringer, David; Le Gros, Graham; Harris, Nicola; Maizels, Rick M.

    2015-01-01

    Over 25% of the world's population are infected with helminth parasites, the majority of which colonise the gastrointestinal tract. However, no vaccine is yet available for human use, and mechanisms of protective immunity remain unclear. In the mouse model of Heligmosomoides polygyrus infection, vaccination with excretory-secretory (HES) antigens from adult parasites elicits sterilising immunity. Notably, three purified HES antigens (VAL-1, -2 and -3) are sufficient for effective vaccination. Protection is fully dependent upon specific IgG1 antibodies, but passive transfer confers only partial immunity to infection, indicating that cellular components are also required. Moreover, immune mice show greater cellular infiltration associated with trapping of larvae in the gut wall prior to their maturation. Intra-vital imaging of infected intestinal tissue revealed a four-fold increase in extravasation by LysM+GFP+ myeloid cells in vaccinated mice, and the massing of these cells around immature larvae. Mice deficient in FcRγ chain or C3 complement component remain fully immune, suggesting that in the presence of antibodies that directly neutralise parasite molecules, the myeloid compartment may attack larvae more quickly and effectively. Immunity to challenge infection was compromised in IL-4Rα- and IL-25-deficient mice, despite levels of specific antibody comparable to immune wild-type controls, while deficiencies in basophils, eosinophils or mast cells or CCR2-dependent inflammatory monocytes did not diminish immunity. Finally, we identify a suite of previously uncharacterised heat-labile vaccine antigens with homologs in human and veterinary parasites that together promote full immunity. Taken together, these data indicate that vaccine-induced immunity to intestinal helminths involves IgG1 antibodies directed against secreted proteins acting in concert with IL-25-dependent Type 2 myeloid effector populations. PMID:25816012

  10. Diagnostic Accuracy of Detecting Hashimoto's Thyroiditis in Thyroid Cancer Patients Who Underwent Thyroid Surgery: Comparison of Ultrasonography, Positron Emission Tomography/CT, Contrast Enhanced CT, and Anti-Thyroid Antibody

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Young Gyun; Lee, Tae Hyun; Park, Dong Hee; Nam, Sang Been [Dept. of Radiology, Korea Cancer Center Hospital, Seoul (Korea, Republic of)

    2012-11-15

    To compare the diagnostic accuracy of ultrasonography (US), F18-fluorodeoxyglucose positron emission tomography/CT (PET/CT), contrast enhanced CT (CECT), serum anti-thyroid antibody for detecting Hashimoto's thyroiditis in thyroid cancer patients who underwent neck surgery. A total of 150 patients with suspicious for thyroid cancer, who had previously undergone US guided needle aspiration of thyroid, were evaluated with the use of US, PET/CT, CECT and serum anti-thyroid antibody. The four studies were performed within two months before neck surgery. Hashimoto's thyroiditis was confirmed by histopathological results. The diagnostic accuracy of US, PET/CT, CECT and serum anti-thyroid antibody were calculated statistically. Hashimoto's thyroiditis was diagnosed in 51 out of the 150 patients, following neck surgery. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of US were 76.5%, 92.9%, 84.8%, 88.5%, and 87.3%, respectively. The corresponding values of PET/CT were 37.3%, 96.0%, 82.6%, 74.8%, and 76.0%, and CECT were 62.7%, 89.9%, 76.2%, 82.4%, and 80.7%, and serum anti-thyroid antibody level were 90.2%, 93.9%, 88.5%, 94.9%, and 92.7%, respectively. McNemar test revealed significant difference among PET/CT and others, but no significant differences among US, CECT and serum anti-thyroid antibody. Overall, serum anti-thyroid antibody showed most accurate diagnostic performance. In detecting Hashimoto's thyroiditis, serum anti-thyroid antibody showed higher diagnostic accuracy than others. US also showed relatively high diagnostic accuracy.

  11. Cloning of Single-Chain Antibody Variants by Overlap-Extension PCR for Evaluation of Antibody Expression in Transient Gene Expression.

    Science.gov (United States)

    Mayrhofer, Patrick; Kunert, Renate

    2017-01-01

    Single-chain fragment variable-fragment crystallizable antibody constructs (scFv-Fc) are homodimeric proteins representing valuable alternatives to heterotetrameric full-length IgG molecules to study protein properties and product-dependent cellular behavior. In contrast to naturally occurring antibodies, these artificial molecules are assembled from functional antibody domains to reduce molecule complexity and enhance antibody expression levels. The scFv-Fc format retains critical antibody functions such as antigen binding affinity and antibody effector functions. Here, we present a protocol to convert the full-length anti-HIV-1 IgG1 antibody 2F5 into a scFv-Fc construct. Variable and constant regions are amplified by conventional PCR reactions and assembled by a single overlap-extension PCR reaction. The amplified product is then cloned into a mammalian expression vector suitable for high-titer transient gene expression. This workflow can be applied to any antibody sequence by adapting the specific primer sequences to the antibody of choice.

  12. Detection of the immunologic rejection after xeno-islet transplantation: a study by MR imaging enhanced with superparamagnetic iron oxide marking CD4+ T cell antibody

    International Nuclear Information System (INIS)

    Nie Wei; Tang Yiya; Rong Pengfei; Ye Bin; Ye Zheng; Tong Qiongjuan; Wang Wei

    2008-01-01

    Objective: To evaluate the feasibility of the diagnosis of the early immunologic rejection after xeno-islet transplantation by MR imaging enhanced with superparamagnetic iron oxide (SPIO) marking CD4 + T cell antibody. Methods: Two thousand neonatal porcine islets (NPI)were transplanted under the left renal capsule of BALB/C nude mice. When the grafts could be observed by MRI, 10 7 human PBMC was intraperitoneal injected to nude mouse models to reconstitute the human immunologic system, 20 mice were reconstituted. Before and 3,7,14 days after reconstitution of human immunologic system on BALB/C nude mice, MRI imaging was performed half an hour after intravenous injection of nano-immunomagnetic beads via vena caudatis to observe the grafts' MRI signal. BALB/C nude mice were sacrificed after MRI scanning immediately, the histopathologic examination was assessed on grafts, the results were compared with MRI results. And calculate the sensitivity, specificity, Youden index number and coincidence of the MRI for immunologic rejection. Results: Grafts can be observed by MRI 3 weeks after islet cell transplantation (before immunologic rejection modeling), there is no abnormal MRI signal detected in nude mice' graft region after microbeads injected. Seven days after building of immunologic rejection model, MRI hypo-signal in graft site is shown in the T 2 WI sequence after nano-bioprober injected. Histopathologic assessments were employed on grafts in nude mice immediately (HE and immunohistochemistry staining), the results shown that there are a lot of T lymphocyts infiltrated in graft region, implying the occurrence of immunologic rejection. And the sensitivity, specificity, Youden index number and coincidence is: (72.96±0.24)%, 100%, 0.73±0.24, (88.46±0.13)% respectively. The correct Kappa between the MRI and the imunohistochemistry staining was 0.76. Conclusion: The cellular immunological rejection to xeno-islet grarts can be assessed with nano-bioprobe with anti-CD4

  13. Polo-like kinase inhibitor volasertib marginally enhances the efficacy of the novel Fc-engineered anti-CD33 antibody BI 836858 in acute myeloid leukemia

    Science.gov (United States)

    Gopalakrishnan, Bhavani; Cheney, Carolyn; Mani, Rajeswaran; Mo, Xiaokui; Bucci, Donna; Walker, Alison; Klisovic, Rebecca; Bhatnagar, Bhavana; Walsh, Katherine; Rueter, Bjoern; Waizenegger, Irene C.; Heider, Karl-Heinz; Blum, William; Vasu, Sumithira; Muthusamy, Natarajan

    2018-01-01

    Acute myeloid leukemia (AML) is the second most common type of leukemia in adults. Incidence of AML increases with age with a peak incidence at 67 years. Patients older than 60 years have an unfavorable prognosis due to resistance to conventional chemotherapy. Volasertib (BI 6727) is a cell-cycle regulator targeting polo-like kinase which has been evaluated in clinical trials in AML. We evaluated effects of volasertib in primary patient samples and NK cells. At equivalent doses, volasertib is cytotoxic to AML blasts but largely spares healthy NK cells. We then evaluated the effect of volasertib treatment in combination with BI 836858 on primary AML blast samples using antibody-dependent cellular cytotoxicity (ADCC) assays. Volasertib treatment of NK cells did not impair NK function as evidenced by comparable levels of BI 836858 mediated ADCC in both volasertib-treated and control-treated NK cells. In summary, volasertib is cytotoxic to AML blasts while sparing NK cell viability and function. Higher BI 836858 mediated ADCC was observed in patient samples pretreated with volasertib. These findings provide a strong rationale to test combination of BI 836858 and volasertib in AML. PMID:29515764

  14. HIV-1 envelope glycoprotein resistance to monoclonal antibody 2G12 is subject-specific and context-dependent in macaques and humans

    NARCIS (Netherlands)

    Malherbe, Delphine C.; Sanders, Rogier W.; van Gils, Marit J.; Park, Byung; Gomes, Michelle M.; Schuitemaker, Hanneke; Barnett, Susan; Haigwood, Nancy L.

    2013-01-01

    HIV-1 Envelope (Env) protein is the sole target of neutralizing antibodies (NAbs) that arise during infection to neutralize autologous variants. Under this immune pressure, HIV escape variants are continuously selected and over the course of infection Env becomes more neutralization resistant. Many

  15. Broadly neutralizing HIV antibodies define a glycan-dependent epitope on the prefusion conformation of gp41 on cleaved envelope trimers

    NARCIS (Netherlands)

    Falkowska, Emilia; Le, Khoa M.; Ramos, Alejandra; Doores, Katie J.; Lee, Jeong Hyun; Blattner, Claudia; Ramirez, Alejandro; Derking, Ronald; van Gils, Marit J.; Liang, Chi-Hui; McBride, Ryan; von Bredow, Benjamin; Shivatare, Sachin S.; Wu, Chung-Yi; Chan-Hui, Po-Ying; Liu, Yan; Feizi, Ten; Zwick, Michael B.; Koff, Wayne C.; Seaman, Michael S.; Swiderek, Kristine; Moore, John P.; Evans, David; Paulson, James C.; Wong, Chi-Huey; Ward, Andrew B.; Wilson, Ian A.; Sanders, Rogier W.; Poignard, Pascal; Burton, Dennis R.

    2014-01-01

    Broadly neutralizing HIV antibodies are much sought after (a) to guide vaccine design, both as templates and as indicators of the authenticity of vaccine candidates, (b) to assist in structural studies, and (c) to serve as potential therapeutics. However, the number of targets on the viral envelope

  16. Differences in human antibody reactivity to Plasmodium falciparum variant surface antigens are dependent on age and malaria transmission intensity in northeastern Tanzania

    DEFF Research Database (Denmark)

    Vestergaard, Lasse S; Lusingu, John P; Nielsen, Morten A

    2008-01-01

    at population level, we conducted an immunoepidemiological study in nearby communities in northeastern Tanzania, situated at different altitudes and therefore exposed to different levels of P. falciparum transmission intensity. Samples of plasma and infected red blood cells (IRBC) were collected from 759...... and functional characteristics of the variant-specific antibody response, which is likely to be important for protection against malaria....

  17. Task-dependent enhancement of facial expression and identity representations in human cortex.

    Science.gov (United States)

    Dobs, Katharina; Schultz, Johannes; Bülthoff, Isabelle; Gardner, Justin L

    2018-02-10

    What cortical mechanisms allow humans to easily discern the expression or identity of a face? Subjects detected changes in expression or identity of a stream of dynamic faces while we measured BOLD responses from topographically and functionally defined areas throughout the visual hierarchy. Responses in dorsal areas increased during the expression task, whereas responses in ventral areas increased during the identity task, consistent with previous studies. Similar to ventral areas, early visual areas showed increased activity during the identity task. If visual responses are weighted by perceptual mechanisms according to their magnitude, these increased responses would lead to improved attentional selection of the task-appropriate facial aspect. Alternatively, increased responses could be a signature of a sensitivity enhancement mechanism that improves representations of the attended facial aspect. Consistent with the latter sensitivity enhancement mechanism, attending to expression led to enhanced decoding of exemplars of expression both in early visual and dorsal areas relative to attending identity. Similarly, decoding identity exemplars when attending to identity was improved in dorsal and ventral areas. We conclude that attending to expression or identity of dynamic faces is associated with increased selectivity in representations consistent with sensitivity enhancement. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  18. Musicians demonstrate experience-dependent brainstem enhancement of musical scale features within continuously gliding pitch.

    Science.gov (United States)

    Bidelman, Gavin M; Gandour, Jackson T; Krishnan, Ananthanarayan

    2011-10-10

    In contrast to language, where pitch patterns consist of continuous and curvilinear contours, musical pitch consists of relatively discrete, stair-stepped sequences of notes. Behavioral and neurophysiological studies suggest that both tone-language and music experience enhance the representation of pitch cues associated with a listener's domain of expertise, e.g., curvilinear pitch in language, discrete scale steps in music. We compared brainstem frequency-following responses (FFRs) of English-speaking musicians (musical pitch experience) and native speakers of Mandarin Chinese (linguistic pitch experience) elicited by rising and falling tonal sweeps that are exemplary of Mandarin tonal contours but uncharacteristic of the pitch patterns typically found in music. In spite of musicians' unfamiliarity with such glides, we find that their brainstem FFRs show enhancement of the stimulus where the curvilinear sweep traverses discrete notes along the diatonic musical scale. This enhancement was note specific in that it was not observed immediately preceding or following the scale tone of interest (passing note). No such enhancements were observed in Chinese listeners. These findings suggest that the musician's brainstem may be differentially tuned by long-term exposure to the pitch patterns inherent to music, extracting pitch in relation to a fixed, hierarchical scale. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  19. Measurement bias dependence of enhanced bipolar gain degradation at low dose rates

    International Nuclear Information System (INIS)

    Witczak, S.C.; Lacoe, R.C.; Mayer, D.C.; Fleetwood, D.M.

    1998-03-01

    Oxide trapped charge, field effects from emitter metallization, and high level injection phenomena moderate enhanced gain degradation of lateral pnp transistors at low dose rates. Hardness assurance tests at elevated irradiation temperatures require larger design margins for low power measurement biases

  20. Effects of MF59 Adjuvant on Induction of Isotype-Switched IgG Antibodies and Protection after Immunization with T-Dependent Influenza Virus Vaccine in the Absence of CD4+ T Cells.

    Science.gov (United States)

    Ko, Eun-Ju; Lee, Young-Tae; Kim, Ki-Hye; Jung, Yu-Jin; Lee, Youri; Denning, Timothy L; Kang, Sang-Moo

    2016-08-01

    CD4(+) T cells play a central role in orchestrating adaptive immunity. To better understand the roles of CD4(+) T cells in the effects of adjuvants, we investigated the efficacy of a T-dependent influenza virus split vaccine with MF59 or alum in CD4 knockout (CD4KO) and wild-type (WT) mice. CD4(+) T cells were required for the induction of IgG antibody responses to the split vaccine and the effects of alum adjuvant. In contrast, MF59 was found to be highly effective in raising isotype-switched IgG antibodies to a T-dependent influenza virus split vaccine in CD4KO mice or CD4-depleted WT mice equivalent to those in intact WT mice, thus overcoming the deficiency of CD4(+) T cells in helping B cells and inducing immunity against influenza virus. Vaccination with the MF59-adjuvanted influenza virus vaccine was able to induce protective CD8(+) T cells and long-lived antibody-secreting cells in CD4KO mice. The effects of MF59 adjuvant in CD4KO mice might be associated with uric acid, inflammatory cytokines, and the recruitment of multiple immune cells at the injection site, but their cellularity and phenotypes were different from those in WT mice. These findings suggest a new paradigm of CD4-independent adjuvant mechanisms, providing the rationales to improve vaccine efficacy in infants, the elderly, immunocompromised patients, as well as healthy adults. MF59-adjuvanted influenza vaccines were licensed for human vaccination, but the detailed mechanisms are not fully elucidated. CD4(+) T cells are required to induce antibody isotype switching and long-term memory responses. In contrast, we discovered that MF59 was highly effective in inducing isotype-switched IgG antibodies and long-term protective immune responses to a T-dependent influenza vaccine independent of CD4(+) T cells. These findings are highly significant for the following reasons: (i) MF59 can overcome a defect of CD4(+) T cells in inducing protective immunity to vaccination with a T-dependent influenza virus

  1. PARP-1 enhances the mismatch-dependence of 5′-directed excision in human mismatch repair in vitro

    Science.gov (United States)

    Liu, Yiyong; Kadyrov, Farid A.; Modrich, Paul

    2011-01-01

    End-directed mismatch-provoked excision has been reconstituted in several purified systems. While 3′-directed excision displays a mismatch dependence similar to that observed in nuclear extracts (≈ 20-fold), the mismatch dependence of 5′-directed excision is only 3 to 4-fold, significantly less than that in extracts (8 to 10-fold). Utilizing a fractionation-based approach, we have isolated a single polypeptide that enhances mismatch dependence of reconstituted 5′-directed excision and have shown it to be identical to poly[ADP-ribose] polymerase 1 (PARP-1). Titration of reconstituted excision reactions or PARP-1-depleted HeLa nuclear extract with purified PARP-1 showed that the protein specifically enhances mismatch dependence of 5′-directed excision. Analysis of a set of PARP-1 mutants revealed that the DNA binding domain and BRCT fold contribute to the regulation of excision specificity. Involvement of the catalytic domain is restricted to its ability to poly(ADP-ribosyl)ate PARP-1 in the presence of NAD+, likely through interference with DNA binding. Analysis of protein-protein interactions demonstrated that PARP-1 interacts with mismatch repair proteins MutSα, exonuclease 1, replication protein A (RPA), and as previously shown by others, replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) as well. The BRCT fold plays an important role in the interaction of PARP-1 with the former three proteins. PMID:21945626

  2. Hydrogen-enhanced fatigue crack growth in steels and its frequency dependence

    Science.gov (United States)

    Matsunaga, Hisao; Takakuwa, Osamu; Yamabe, Junichiro; Matsuoka, Saburo

    2017-06-01

    In the context of the fatigue life design of components, particularly those destined for use in hydrogen refuelling stations and fuel cell vehicles, it is important to understand the hydrogen-induced, fatigue crack growth (FCG) acceleration in steels. As such, the mechanisms for acceleration and its influencing factors are reviewed and discussed in this paper, with a special focus on the peculiar frequency dependence of the hydrogen-induced FCG acceleration. Further, this frequency dependence is debated by introducing some potentially responsible elements, along with new experimental data obtained by the authors. This article is part of the themed issue 'The challenges of hydrogen and metals'.

  3. Lauric acid dependent enhancement in hepatic SCPx protein requires an insulin deficient environment.

    Science.gov (United States)

    Lopez, Dayami; Niesen, Melissa; Bedi, Mohini; McLean, Mark P

    2008-02-01

    Sterol carrier protein X (SCPx) is a peroxisomal protein with both lipid transfer and thiolase activity. Treating with the fatty acid, lauric acid, induced SCPx mRNA levels in rat liver and in rat hepatoma H4IIE cells but enhanced protein levels of SCPx and the thiolase produced as a post-translational modification of SCPx were only seen in H4IIE cells. Further investigation revealed that the presence of insulin can mask lauric acid effects on the SCPx gene especially at the protein level. These data are in agreement with the findings that diabetes, a medical condition characterized by high levels of fatty acids in an insulin deficient environment, enhances the hepatic expression of SCPx.

  4. Depth enhancement of multi-layer light field display using polarization dependent internal reflection.

    Science.gov (United States)

    Jo, Na-Young; Lim, Hong-Gi; Lee, Sung-Keun; Kim, Yong-Soo; Park, Jae-Hyeung

    2013-12-02

    A technique to enhance the depth range of the multi-layer light field three-dimensional display is proposed. A set of the optical plates are stacked in front of the conventional multi-layer light field display, creating additional internal reflection for one polarization state. By switching between two orthogonal polarization states in synchronization with the displayed three-dimensional images, the depth range of the display can be doubled. The proposed method is verified experimentally, confirming its feasibility.

  5. Feature-size dependent selective edge enhancement of x-ray images

    International Nuclear Information System (INIS)

    Herman, S.

    1988-01-01

    Morphological filters are nonlinear signal transformations that operate on a picture directly in the space domain. Such filters are based on the theory of mathematical morphology previously formulated. The filt4er being presented here features a ''mask'' operator (called a ''structuring element'' in some of the literature) which is a function of the two spatial coordinates x and y. The two basic mathematical operations are called ''masked erosion'' and ''masked dilation''. In the case of masked erosion the mask is passed over the input image in a raster pattern. At each position of the mask, the pixel values under the mask are multiplied by the mask pixel values. Then the output pixel value, located at the center position of the mask,is set equal to the minimum of the product of the mask and input values. Similarity, for masked dilation, the output pixel value is the maximum of the product of the input and the mask pixel values. The two basic processes of dilation and erosion can be used to construct the next level of operations the ''positive sieve'' (also called ''opening'') and the ''negative sieve'' (''closing''). The positive sieve modifies the peaks in the image whereas the negative sieve works on image valleys. The positive sieve is implemented by passing the output of the masked erosion step through the masked dilation function. The negative sieve reverses this procedure, using a dilation followed by an erosion. Each such sifting operator is characterized by a ''hole size''. It will be shown that the choice of hole size will select the range of pixel detail sizes which are to be enhanced. The shape of the mask will govern the shape of the enhancement. Finally positive sifting is used to enhance positive-going (peak) features, whereas negative enhances the negative-going (valley) landmarks

  6. Overexpression of the cochaperone CHIP enhances Hsp70-dependent folding activity in mammalian cells

    NARCIS (Netherlands)

    Kampinga, HH; Kanon, B; Salomons, FA; Kabakov, AE; Patterson, C

    CHIP is a cochaperone of Hsp70 that inhibits Hsp70-dependent refolding in vitro. However, the effect of altered expression of CHIP on the fate of unfolded proteins in mammalian cells has not been determined. Surprisingly, we found that overexpression of CHIP in fibroblasts increased the refolding of

  7. Randomized Trial of Continuing Care Enhancements for Cocaine-Dependent Patients following Initial Engagement

    Science.gov (United States)

    McKay, James R.; Lynch, Kevin G.; Coviello, Donna; Morrison, Rebecca; Cary, Mark S.; Skalina, Lauren; Plebani, Jennifer

    2010-01-01

    Objective: The effects of cognitive-behavioral relapse prevention (RP), contingency management (CM), and their combination (CM + RP) were evaluated in a randomized trial with 100 cocaine-dependent patients (58% female, 89% African American) who were engaged in treatment for at least 2 weeks and had an average of 44 days of abstinence at baseline.…

  8. Arachidonic acid supplementation enhances in vitro skeletal muscle cell growth via a COX-2-dependent pathway.

    Science.gov (United States)

    Markworth, James F; Cameron-Smith, David

    2013-01-01

    Arachidonic acid (AA) is the metabolic precursor to a diverse range of downstream bioactive lipid mediators. A positive or negative influence of individual eicosanoid species [e.g., prostaglandins (PGs), leukotrienes, and hydroxyeicosatetraenoic acids] has been implicated in skeletal muscle cell growth and development. The collective role of AA-derived metabolites in physiological states of skeletal muscle growth/atrophy remains unclear. The present study aimed to determine the direct effect of free AA supplementation and subsequent eicosanoid biosynthesis on skeletal myocyte growth in vitro. C2C12 (mouse) skeletal myocytes induced to differentiate with supplemental AA exhibited dose-dependent increases in the size, myonuclear content, and protein accretion of developing myotubes, independent of changes in cell density or the rate/extent of myogenic differentiation. Nonselective (indomethacin) or cyclooxygenase 2 (COX-2)-selective (NS-398) nonsteroidal anti-inflammatory drugs blunted basal myogenesis, an effect that was amplified in the presence of supplemental free AA substrate. The stimulatory effects of AA persisted in preexisting myotubes via a COX-2-dependent (NS-389-sensitive) pathway, specifically implying dependency on downstream PG biosynthesis. AA-stimulated growth was associated with markedly increased secretion of PGF(2α) and PGE(2); however, incubation of myocytes with PG-rich conditioned medium failed to mimic the effects of direct AA supplementation. In vitro AA supplementation stimulates PG release and skeletal muscle cell hypertrophy via a COX-2-dependent pathway.

  9. Hes1 promotes the IL-22-mediated antimicrobial response by enhancing STAT3-dependent transcription in human intestinal epithelial cells

    International Nuclear Information System (INIS)

    Murano, Tatsuro; Okamoto, Ryuichi; Ito, Go; Nakata, Toru; Hibiya, Shuji; Shimizu, Hiromichi; Fujii, Satoru; Kano, Yoshihito; Mizutani, Tomohiro; Yui, Shiro; Akiyama-Morio, Junko; Nemoto, Yasuhiro; Tsuchiya, Kiichiro; Nakamura, Tetsuya; Watanabe, Mamoru

    2014-01-01

    Highlights: •Hes1 enhances IL-22-STAT3 signaling in human intestinal epithelial cells. •Hes1 enhances REG family gene induction by IL-22-STAT3 signaling. •Protein level of Hes1 restricts the response to IL-22. •Present regulation of a cytokine signal represents a new mode of Hes1 function. -- Abstract: Notch signaling plays an essential role in the proliferation and differentiation of intestinal epithelial cells (IECs). We have previously shown that Notch signaling is up-regulated in the inflamed mucosa of ulcerative colitis (UC) and thereby plays an indispensable role in tissue regeneration. Here we show that in addition to Notch signaling, STAT3 signaling is highly activated in the inflamed mucosa of UC. Forced expression of the Notch target gene Hes1 dramatically enhanced the IL-22-mediated STAT3-dependent transcription in human IECs. This enhancement of STAT3-dependent transcription was achieved by the extended phosphorylation of STAT3 by Hes1. Microarray analysis revealed that Hes1-mediated enhancement of IL-22-STAT3 signaling significantly increased the induction of genes encoding antimicrobial peptides, such as REG1A, REG3A and REG3G, in human IECs. Conversely, the reduction of Hes1 protein levels with a γ-secretase inhibitor significantly down-regulated the induction of those genes in IECs, resulting in a markedly poor response to IL-22. Our present findings identify a new role for the molecular function of Hes1 in which the protein can interact with cytokine signals and regulate the immune response of IECs

  10. Hes1 promotes the IL-22-mediated antimicrobial response by enhancing STAT3-dependent transcription in human intestinal epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Murano, Tatsuro [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Okamoto, Ryuichi, E-mail: rokamoto.gast@tmd.ac.jp [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Department of Advanced GI Therapeutics, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Ito, Go; Nakata, Toru; Hibiya, Shuji; Shimizu, Hiromichi; Fujii, Satoru; Kano, Yoshihito; Mizutani, Tomohiro; Yui, Shiro; Akiyama-Morio, Junko; Nemoto, Yasuhiro [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Tsuchiya, Kiichiro; Nakamura, Tetsuya [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Department of Advanced GI Therapeutics, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Watanabe, Mamoru [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan)

    2014-01-17

    Highlights: •Hes1 enhances IL-22-STAT3 signaling in human intestinal epithelial cells. •Hes1 enhances REG family gene induction by IL-22-STAT3 signaling. •Protein level of Hes1 restricts the response to IL-22. •Present regulation of a cytokine signal represents a new mode of Hes1 function. -- Abstract: Notch signaling plays an essential role in the proliferation and differentiation of intestinal epithelial cells (IECs). We have previously shown that Notch signaling is up-regulated in the inflamed mucosa of ulcerative colitis (UC) and thereby plays an indispensable role in tissue regeneration. Here we show that in addition to Notch signaling, STAT3 signaling is highly activated in the inflamed mucosa of UC. Forced expression of the Notch target gene Hes1 dramatically enhanced the IL-22-mediated STAT3-dependent transcription in human IECs. This enhancement of STAT3-dependent transcription was achieved by the extended phosphorylation of STAT3 by Hes1. Microarray analysis revealed that Hes1-mediated enhancement of IL-22-STAT3 signaling significantly increased the induction of genes encoding antimicrobial peptides, such as REG1A, REG3A and REG3G, in human IECs. Conversely, the reduction of Hes1 protein levels with a γ-secretase inhibitor significantly down-regulated the induction of those genes in IECs, resulting in a markedly poor response to IL-22. Our present findings identify a new role for the molecular function of Hes1 in which the protein can interact with cytokine signals and regulate the immune response of IECs.

  11. Comparative analysis of Luminex-based donor-specific antibody mean fluorescence intensity values with complement-dependent cytotoxicity & flow crossmatch results in live donor renal transplantation

    OpenAIRE

    Ajay Kumar Baranwal; Deepali Krishan Bhat; Sanjeev Goswami; Sanjay Kumar Agarwal; Gurvinder Kaur; Jasmeet Kaur; Narinder Mehra

    2017-01-01

    Background & objectives: Antibodies specific to donor human leucocyte antigen (HLA) play a critical role in graft rejection and graft loss. In recent years, techniques for their detection have evolved significantly providing an ever-increasing degree of sensitivity and specificity, from the conventional cell-based assays to the advanced solid-phase system based on the Luminex platform. Consensus is still evolving on the routine employment of all these methods, either stand alone or in combina...

  12. Enhancing the sensitivity of immunoassay procedures by use of antibodies directed to the product of a reaction between probe labels and assay substrates

    Science.gov (United States)

    Erlanger, B.F.; Chen, B.X.

    1997-07-22

    The subject invention provides an antibody which specifically binds to the product of a reaction between a labeling substance and a substrate. The subject invention also provides a method of making an immunogen used to produce the antibody of the subject invention. The invention further provides methods of using the subject antibody for detecting an antigen of interest in a sample, for example detecting a protein comprising an amino acid sequence of interest and detecting a nucleic acid molecule comprising a nucleic acid sequence of interest. 8 figs.

  13. Momentum and zenithal dependence of the enhancements of intensities of cosmic ray muons

    International Nuclear Information System (INIS)

    Abdel-Monem, M.S.; Osborne, A.R.; Benbrook, J.R.; Sheldon, W.R.; Duller, N.M.; Green, P.J.; Choate, L.M.; Magnusson, C.E.

    1977-01-01

    The absolute directional differential intensities of high-energy cosmic ray muons near sea level have been measured over the momentum range 2-700 GeV/c in the vertical direction and zenithal interval 55deg-90deg. The measurements were made with the AMH magnetic spectrometer-telescope. The enhancements I(65deg)/I(0deg) and I(80deg)/I(0deg) of the muon intensities as a function of momentum are presented and compared with the theoretical results of Maeda and Asbury et al. (author)

  14. Temperature dependence of the surface enhanced raman spectroelectrochemistry of iron in aqueous solutions.

    Energy Technology Data Exchange (ETDEWEB)

    Simpson, L. J.; Melendres, C. A.; Chemical Engineering

    1996-06-01

    The effect of temperature on the composition of the corrosion film on iron in aqueous sodium hydroxide and borate solutions was investigated using surface enhanced Raman spectroelectrochemistry (SERS). Fe(OH){sub 2} and Fe{sub 3}O{sub 4} were observed in the prepassivation region, while Fe{sub 3}O{sub 4} and FeOOH accounted for most of the passivated film at 25, 60 and 95 C. Fe(OH){sub 2} was found to be a stable component of the corrosion film on iron at 95 C, which is contrary to recently published theoretical calculations.

  15. Determining the Phagocytic Activity of Clinical Antibody Samples

    Science.gov (United States)

    McAndrew, Elizabeth G.; Dugast, Anne-Sophie; Licht, Anna F.; Eusebio, Justin R.; Alter, Galit; Ackerman, Margaret E.

    2011-01-01

    Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3. Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome. Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8. Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 μm fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple FcγRs, including both inhibitory and activating. Assay output can

  16. TMR-MCDB: Enhancing Security in a Multi-cloud Model through Improvement of Service Dependability

    OpenAIRE

    Mohammed Alzain; Ben Soh; Eric Pardede

    2014-01-01

    In IT enterprises, different computing needs are provided as a service. The service providers take care of the customers’ needs by, for example, maintaining software or purchasing expensive hardware. In addition, there are many benefits of using the technology available from cloud service providers, such as access to large-scale, on-demand, flexible computing infrastructures. However, increasing the dependability of cloud computing is important in order for its potential to be realized. Data ...

  17. Time- but not sleep-dependent consolidation of tDCS-enhanced visuomotor skills.

    Science.gov (United States)

    Reis, Janine; Fischer, Jan Torben; Prichard, George; Weiller, Cornelius; Cohen, Leonardo G; Fritsch, Brita

    2015-01-01

    Consolidation of motor skills after training can occur in a time- or sleep-dependent fashion. Recent studies revealed time-dependent consolidation as a common feature of visuomotor tasks. We have previously shown that anodal transcranial direct current stimulation (tDCS) in combination with repeated motor training benefits consolidation by the induction of offline skill gains in a complex visuomotor task, preventing the regular occurrence of skill loss between days. Here, we asked 2 questions: What is the time course of consolidation between days for this task and do exogenously induced offline gains develop as a function of time or overnight sleep? We found that both the development of offline skill loss in sham-stimulated subjects and offline skill gains induced by anodal tDCS critically depend on the passage of time after training, but not on overnight sleep. These findings support the view that tDCS interacts directly with the physiological consolidation process. However, in a control experiment, anodal tDCS applied after the training did not induce skill gains, implying that coapplication of tDCS and training is required to induce offline skill gains, pointing to the initiation of consolidation already during training. Published by Oxford University Press 2013. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  18. Frequency-Dependent Amplitude Panning for the Stereophonic Image Enhancement of Audio Recorded Using Two Closely Spaced Microphones

    Directory of Open Access Journals (Sweden)

    Chan Jun Chun

    2016-02-01

    Full Text Available In this paper, we propose a new frequency-dependent amplitude panning method for stereophonic image enhancement applied to a sound source recorded using two closely spaced omni-directional microphones. The ability to detect the direction of such a sound source is limited due to weak spatial information, such as the inter-channel time difference (ICTD and inter-channel level difference (ICLD. Moreover, when sound sources are recorded in a convolutive or a real room environment, the detection of sources is affected by reverberation effects. Thus, the proposed method first tries to estimate the source direction depending on the frequency using azimuth-frequency analysis. Then, a frequency-dependent amplitude panning technique is proposed to enhance the stereophonic image by modifying the stereophonic law of sines. To demonstrate the effectiveness of the proposed method, we compare its performance with that of a conventional method based on the beamforming technique in terms of directivity pattern, perceived direction, and quality degradation under three different recording conditions (anechoic, convolutive, and real reverberant. The comparison shows that the proposed method gives us better stereophonic images in a stereo loudspeaker reproduction than the conventional method without any annoying effects.

  19. Frequency-Dependent Enhancement of Fluid Intelligence Induced by Transcranial Oscillatory Potentials

    Energy Technology Data Exchange (ETDEWEB)

    Santarnecchi, Emiliano [Univ. of Siena (Italy). Dept. of Medicine, Surgery and Neuroscience; Polizzotto, Nicola Riccardo [Univ. of Pittsburgh, PA (United States). Dept. of Psychiatry; Godone, Marco [Univ. of Siena (Italy). Dept. of Medicine, Surgery and Neuroscience; Giovannelli, Fabio [San Giovanni di Dio Hospital, Florence (Italy). Complex Unit of Neurology; Feurra, Matteo [Univ. of Siena (Italy). Dept. of Medicine, Surgery and Neuroscience; Matzen, Laura [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Rossi, Alessandro [Univ. of Siena (Italy). Dept. of Medicine, Surgery and Neuroscience; Rossi, Simone [Univ. of Siena (Italy). Dept. of Medicine, Surgery and Neuroscience

    2013-08-05

    Everyday problem solving requires the ability to go beyond experience by efficiently encoding and manipulating new information, i.e., fluid intelligence (Gf) [1]. Performance in tasks involving Gf, such as logical and abstract reasoning, has been shown to rely on distributed neural networks, with a crucial role played by prefrontal regions [2]. Synchronization of neuronal activity in the gamma band is a ubiquitous phenomenon within the brain; however, no evidence of its causal involvement in cognition exists to date [3]. Here, we show an enhancement of Gf ability in a cognitive task induced by exogenous rhythmic stimulation within the gamma band. Imperceptible alternating current [4] delivered through the scalp over the left middle frontal gyrus resulted in a frequency-specific shortening of the time required to find the correct solution in a visuospatial abstract reasoning task classically employed to measure Gf abilities (i.e., Raven’s matrices) [5]. Crucially, gamma-band stimulation (γ-tACS) selectively enhanced performance only on more complex trials involving conditional/logical reasoning. The finding presented here supports a direct involvement of gamma oscillatory activity in the mechanisms underlying higher-order human cognition.

  20. Musicians have enhanced audiovisual multisensory binding: experience-dependent effects in the double-flash illusion.

    Science.gov (United States)

    Bidelman, Gavin M

    2016-10-01

    Musical training is associated with behavioral and neurophysiological enhancements in auditory processing for both musical and nonmusical sounds (e.g., speech). Yet, whether the benefits of musicianship extend beyond enhancements to auditory-specific skills and impact multisensory (e.g., audiovisual) processing has yet to be fully validated. Here, we investigated multisensory integration of auditory and visual information in musicians and nonmusicians using a double-flash illusion, whereby the presentation of multiple auditory stimuli (beeps) concurrent with a single visual object (flash) induces an illusory perception of multiple flashes. We parametrically varied the onset asynchrony between auditory and visual events (leads and lags of ±300 ms) to quantify participants' "temporal window" of integration, i.e., stimuli in which auditory and visual cues were fused into a single percept. Results show that musically trained individuals were both faster and more accurate at processing concurrent audiovisual cues than their nonmusician peers; nonmusicians had a higher susceptibility for responding to audiovisual illusions and perceived double flashes over an extended range of onset asynchronies compared to trained musicians. Moreover, temporal window estimates indicated that musicians' windows (listening skills, improving multimodal processing and the integration of multiple sensory systems in a domain-general manner.

  1. Exercise training enhances baroreflex sensitivity by an angiotensin II-dependent mechanism in chronic heart failure.

    Science.gov (United States)

    Mousa, Tarek M; Liu, Dongmei; Cornish, Kurtis G; Zucker, Irving H

    2008-03-01

    Exercise training (EX) has become an important modality capable of enhancing the quality of life and survival of patients with chronic heart failure (CHF). Although 4 wk of EX in animals with CHF evoked a reduction in renal sympathetic nerve activity and ANG II plasma levels and an enhancement in baroreflex sensitivity at rest (Liu JL, Irvine S, Reid IA, Patel KP, Zucker IH, Circulation 102: 1854-1862, 2000; Liu JL, Kulakofsky J, Zucker IH, J Appl Physiol 92: 2403-2408, 2002), it is unclear whether these phenomena are causally related. CHF was induced in rabbits by ventricular pacing (360-380 beats/min) for 3 wk. CHF rabbits were EX for 4 wk at 15-18 m/min, 6 days/wk, 30-40 min/day. Three groups of rabbits were studied: CHF (with no EX), CHF-EX, and CHF-EX + ANG II infusion [in which ANG II levels were kept at or near levels observed in CHF (non-EX) rabbits by subcutaneous osmotic minipump infusion]. EX prevented the increase in plasma ANG II levels shown in CHF rabbits. CHF and CHF-EX + ANG II infusion rabbits had significantly depressed baroreflex sensitivity slopes (P baroreflex function in CHF after EX are due to the concomitant reduction in ANG II and angiotensin receptors in the central nervous system.

  2. APOBEC3G enhances lymphoma cell radioresistance by promoting cytidine deaminase-dependent DNA repair.

    Science.gov (United States)

    Nowarski, Roni; Wilner, Ofer I; Cheshin, Ori; Shahar, Or D; Kenig, Edan; Baraz, Leah; Britan-Rosich, Elena; Nagler, Arnon; Harris, Reuben S; Goldberg, Michal; Willner, Itamar; Kotler, Moshe

    2012-07-12

    APOBEC3 proteins catalyze deamination of cytidines in single-stranded DNA (ssDNA), providing innate protection against retroviral replication by inducing deleterious dC > dU hypermutation of replication intermediates. APOBEC3G expression is induced in mitogen-activated lymphocytes; however, no physiologic role related to lymphoid cell proliferation has yet to be determined. Moreover, whether APOBEC3G cytidine deaminase activity transcends to processing cellular genomic DNA is unknown. Here we show that lymphoma cells expressing high APOBEC3G levels display efficient repair of genomic DNA double-strand breaks (DSBs) induced by ionizing radiation and enhanced survival of irradiated cells. APOBEC3G transiently accumulated in the nucleus in response to ionizing radiation and was recruited to DSB repair foci. Consistent with a direct role in DSB repair, inhibition of APOBEC3G expression or deaminase activity resulted in deficient DSB repair, whereas reconstitution of APOBEC3G expression in leukemia cells enhanced DSB repair. APOBEC3G activity involved processing of DNA flanking a DSB in an integrated reporter cassette. Atomic force microscopy indicated that APOBEC3G multimers associate with ssDNA termini, triggering multimer disassembly to multiple catalytic units. These results identify APOBEC3G as a prosurvival factor in lymphoma cells, marking APOBEC3G as a potential target for sensitizing lymphoma to radiation therapy.

  3. ARA24/Ran enhances the androgen-dependent NH2- and COOH-terminal interaction of the androgen receptor

    International Nuclear Information System (INIS)

    Harada, Naoki; Ohmori, Yuji; Yamaji, Ryoichi; Higashimura, Yasuki; Okamoto, Kazuki; Isohashi, Fumihide; Nakano, Yoshihisa; Inui, Hiroshi

    2008-01-01

    The androgen receptor (AR) acts as an androgen-dependent transcription factor controlling the development of prostate tissue. Upon binding to androgen, AR undergoes a dynamic structural change leading to interaction between the NH 2 - and COOH-terminal regions of AR (N-C interaction). ARA24/Ran, which is a small GTPase, functions as an AR coactivator. Here, we report that ARA24/Ran enhances the N-C interaction of AR. The constitutively GTP- or GDP-bound form of ARA24/Ran repressed the AR N-C interaction. ARA24/Ran did not enhance the transcriptional activities of AR mutants that disrupt the N-C interaction. ARA24/Ran formed an endogenous protein complex with nuclear AR, but not cytoplasmic AR. Unlike SRC-1 with the positive activity for AR N-C interaction, ARA24/Ran did not enhance the transcriptional activity of the COOH-terminal domain-deleted AR mutant that is constitutively localized in the nucleus. These data demonstrate that ARA24/Ran increases AR transactivation by enhancing the AR N-C interaction in the nucleus

  4. Therapeutic antibodies as a treatment option for dengue fever.

    Science.gov (United States)

    Chan, Kuan Rong; Ong, Eugenia Z; Ooi, Eng Eong

    2013-11-01

    Dengue fever is the most prevalent mosquito-borne viral disease globally with about 100 million cases of acute dengue annually. Severe dengue infection can result in a life-threatening illness. In the absence of either a licensed vaccine or antiviral drug against dengue, therapeutic antibodies that neutralize dengue virus (DENV) may serve as an effective medical countermeasure against severe dengue. However, therapeutic antibodies would need to effectively neutralize all four DENV serotypes. It must not induce antibody-dependent enhancement of DENV infection in monocytes/macrophages through Fc gamma receptor (FcγR)-mediated phagocytosis, which is hypothesized to increase the risk of severe dengue. Here, we review the strategies and technologies that can be adopted to develop antibodies for therapeutic applications. We also discuss the mechanism of antibody neutralization in the cells targeted by DENV that express Fc gamma receptor. These studies have provided significant insight toward the use of therapeutic antibodies as a potentially promising bulwark against dengue.

  5. Spike-timing-dependent plasticity enhanced synchronization transitions induced by autapses in adaptive Newman-Watts neuronal networks.

    Science.gov (United States)

    Gong, Yubing; Wang, Baoying; Xie, Huijuan

    2016-12-01

    In this paper, we numerically study the effect of spike-timing-dependent plasticity (STDP) on synchronization transitions induced by autaptic activity in adaptive Newman-Watts Hodgkin-Huxley neuron networks. It is found that synchronization transitions induced by autaptic delay vary with the adjusting rate A p of STDP and become strongest at a certain A p value, and the A p value increases when network randomness or network size increases. It is also found that the synchronization transitions induced by autaptic delay become strongest at a certain network randomness and network size, and the values increase and related synchronization transitions are enhanced when A p increases. These results show that there is optimal STDP that can enhance the synchronization transitions induced by autaptic delay in the adaptive neuronal networks. These findings provide a new insight into the roles of STDP and autapses for the information transmission in neural systems. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  6. Early survival factor deprivation in the olfactory epithelium enhances activity-dependent survival

    Directory of Open Access Journals (Sweden)

    Adrien eFrançois

    2013-12-01

    Full Text Available The neuronal olfactory epithelium undergoes permanent renewal because of environmental aggression. This renewal is partly regulated by factors modulating the level of neuronal apoptosis. Among them, we had previously characterized endothelin as neuroprotective. In this study, we explored the effect of cell survival factor deprivation in the olfactory epithelium by intranasal delivery of endothelin receptors antagonists to rat pups. This treatment induced an overall increase of apoptosis in the olfactory epithelium. The responses to odorants recorded by electroolfactogram were decreased in treated animal, a result consistent with a loss of olfactory sensory neurons (OSNs. However, the treated animal performed better in an olfactory orientation test based on maternal odor compared to non-treated littermates. This improved performance could be due to activity-dependent neuronal survival of OSNs in the context of increased apoptosis level. In order to demonstrate it, we odorized pups with octanal, a known ligand for the rI7 olfactory receptor (Olr226. We quantified the number of OSN expressing rI7 by RT-qPCR and whole mount in situ hybridization. While this number was reduced by the survival factor removal treatment, this reduction was abolished by the presence of its ligand. This improved survival was optimal for low concentration of odorant and was specific for rI7-expressing OSNs. Meanwhile, the number of rI7-expressing OSNs was not affected by the odorization in non-treated littermates; showing that the activity-dependant survival of OSNs did not affect the OSN population during the 10 days of odorization in control conditions. Overall, our study shows that when apoptosis is promoted in the olfactory mucosa, the activity-dependent neuronal plasticity allows faster tuning of the olfactory sensory neuron population towards detection of environmental odorants.

  7. Lectin-Dependent Enhancement of Ebola Virus Infection via Soluble and Transmembrane C-type Lectin Receptors

    Science.gov (United States)

    Lear, Calli; Chen, Li; Yantosca, L. Michael; Scully, Corinne; Sarraju, Ashish; Sokolovska, Anna; Zariffard, M. Reza; Eisen, Damon P.; Mungall, Bruce A.; Kotton, Darrell N.; Omari, Amel; Huang, I-Chueh; Farzan, Michael; Takahashi, Kazue; Stuart, Lynda; Stahl, Gregory L.; Ezekowitz, Alan B.; Spear, Gregory T.; Olinger, Gene G.; Schmidt, Emmett V.; Michelow, Ian C.

    2013-01-01

    Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active

  8. Lectin-dependent enhancement of Ebola virus infection via soluble and transmembrane C-type lectin receptors.

    Directory of Open Access Journals (Sweden)

    Matthew Brudner

    Full Text Available Mannose-binding lectin (MBL is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion

  9. The enhanced cytotoxicity of misonidazole in the thiol depleted state - An oxygen dependent mechanism

    International Nuclear Information System (INIS)

    Tuttle, S.W.; Varnes, M.E.; Donahue, L.; Biaglow, J.E.

    1985-01-01

    Incubating A549 cells in the presence of L-buthionine-S, R-sulfoximine and misonidazole under aerobic conditions results in lowered rates of cell growth and greater cytotoxicity than is seen with either drug alone. The authors previously demonstrated the accumulation of hydrogen peroxide from cells treated with misonidazole following the inhibition of GSH-peroxidase with thiol depleting agents. They hypothesize that the enhancement of misonidazole toxicity by L-BSO results from the increased exposure to hydrogen peroxide, and the possible formation of the highly reactive hydroxyl radical in the presence of trace metals via Fenton chemistry. Support for this hypothesis comes from their observations that addition of radical scavengers (such as SOD and catalase) and nutritional antioxidants (vitamin E) to the culture medium will partially inhibit the cytotoxic effects. Further work is being done to measure the products of reaction of toxic oxygen species with cellular macromolecules, i.e. lipids

  10. Thickness dependence of polydopamine thin films on detection sensitivity of surface plasmon-enhanced fluorescence biosensors

    Science.gov (United States)

    Toma, Mana; Tawa, Keiko

    2018-03-01

    A bioinspired polydopamine (PDA) coating is a good candidate for the rapid and cheap chemical modification of biosensor surfaces. Herein, we report the effect of PDA thickness on the detection sensitivity of a fluorescence biosensor utilizing surface plasmon-enhanced fluorescence. The thickness of PDA films was tuned by the incubation time of the dopamine solution and varied from 1 to 17 nm. The detection sensitivity was evaluated as the limit of detection (LOD) of a fluorescently labelled target analyte by a model immunoassay. The LOD was determined to be 1.6 pM for the thickest PDA film and was improved to 1.0 pM by reducing the thickness to the range from 1 to 5 nm, corresponding to the incubation time of 10 to 60 min. The experimental results indicate that the PDA coating is suitable for the surface functionalization of biosensors in mass production as it does not require precise control of the incubation time.

  11. Leukotriene B4 Enhances NOD2-Dependent Innate Response against Influenza Virus Infection.

    Directory of Open Access Journals (Sweden)

    Manon Le Bel

    Full Text Available Leukotriene B4 (LTB4, a central mediator of inflammation, is well known for its chemoattractant properties on effectors cells of the immune system. LTB4 also has the ability to control microbial infection by improving host innate defenses through the release of antimicrobial peptides and modulation of intracellular Toll-like receptors (TLRs expression in response to agonist challenge. In this report, we provide evidences that LTB4 acts on nucleotide-binging oligomerization domain 2 (NOD2 pathway to enhance immune response against influenza A infection. Infected mice receiving LTB4 show improved survival, lung architecture and reduced lung viral loads as compared to placebo-treated animals. NOD2 and its downstream adaptor protein IPS-1 have been found to be essential for LTB4-mediated effects against IAV infection, as absence of NOD2 or IPS-1 diminished its capacity to control viral infection. Treatment of IAV-infected mice with LTB4 induces an increased activation of IPS-1-IRF3 axis leading to an enhanced production of IFNβ in lungs of infected mice. LTB4 also has the ability to act on the RICK-NF-κB axis since administration of LTB4 to mice challenged with MDP markedly increases the secretion of IL-6 and TNFα in lungs of mice. TAK1 appears to be essential to the action of LTB4 on NOD2 pathway since pretreatment of MEFs with TAK1 inhibitor prior stimulation with IAV or MDP strongly abrogated the potentiating effects of LTB4 on both IFNβ and cytokine secretion. Together, our results demonstrate that LTB4, through its ability to activate TAK1, potentiates both IPS-1 and RICK axis of the NOD2 pathway to improve host innate responses.

  12. SnO2Inverse Opal Composite Film with Low Angle-dependent Structural Color and Enhanced Mechanical Strength.

    Science.gov (United States)

    Liu, Fangfang; Shan, Bin; Zhang, Shufen; Tang, Bingtao

    2018-03-13

    Structural colors are attracting considerable attention for their advantages of environmental friendliness and resistance to fading. However, the weak mechanical strength and intrinsic iridescent color restrict their widespread application. This article describes a SnO 2 inverse opal composite film with low angle-dependent structural color and enhanced mechanical strength. In the present study, a direct template method was used to prepare SnO 2 inverse opals, which were then embedded in polydimethylsiloxane (PDMS). The structural colors of obtained composite films were low angle-dependent due to light scattering and high effective refractive index. Meanwhile, owing to the good physical strength of PDMS, structures of SnO 2 inverse opals were provided with effective protection. No specific wavelength shift occurred during stretching, and exhibited excellent cycling stability. All these advantages indicated potential applications in packing and decorating materials.

  13. One Dose of Staphylococcus aureus 4C-Staph Vaccine Formulated with a Novel TLR7-Dependent Adjuvant Rapidly Protects Mice through Antibodies, Effector CD4+ T Cells, and IL-17A.

    Science.gov (United States)

    Mancini, Francesca; Monaci, Elisabetta; Lofano, Giuseppe; Torre, Antonina; Bacconi, Marta; Tavarini, Simona; Sammicheli, Chiara; Arcidiacono, Letizia; Galletti, Bruno; Laera, Donatello; Pallaoro, Michele; Tuscano, Giovanna; Fontana, Maria Rita; Bensi, Giuliano; Grandi, Guido; Rossi-Paccani, Silvia; Nuti, Sandra; Rappuoli, Rino; De Gregorio, Ennio; Bagnoli, Fabio; Soldaini, Elisabetta; Bertholet, Sylvie

    2016-01-01

    A rapidly acting, single dose vaccine against Staphylococcus aureus would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent S. aureus candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4+ T cells not only reduced survival but also increased S. aureus load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by in vivo neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against S. aureus through the combined actions of antibodies, CD4+ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units.

  14. One Dose of Staphylococcus aureus 4C-Staph Vaccine Formulated with a Novel TLR7-Dependent Adjuvant Rapidly Protects Mice through Antibodies, Effector CD4+ T Cells, and IL-17A.

    Directory of Open Access Journals (Sweden)

    Francesca Mancini

    Full Text Available A rapidly acting, single dose vaccine against Staphylococcus aureus would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent S. aureus candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4+ T cells not only reduced survival but also increased S. aureus load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by in vivo neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against S. aureus through the combined actions of antibodies, CD4+ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units.

  15. Detection of antibodies to co-trimoxazole (preservative drug interfering with routine red cell antibody screening

    Directory of Open Access Journals (Sweden)

    Deepti Sachan

    2018-01-01

    Full Text Available Drug-dependent antibodies can rarely cause interference in pretransfusion antibody screening. The diluents for commercial reagent red blood cells contain different antibiotics, such as chloramphenicol, neomycin sulfate, and gentamycin as a preservative. The presence of antibodies to a given drug in patient may lead to positive results when performing antibody identification. We present a rare case of detection of anti-co-trimoxazole antibody during routine antibody screening in a female patient undergoing neurosurgery. These antibodies mimicked as antibody against high-frequency red cell antigens reacting in both saline phase as well as antiglobulin phase. Anti-co-trimoxazole antibody was confirmed by repeating antibody screen using reagent red cells of different manufacturers with and without co-trimoxazole drug as preservative as well as using washed red cell panels. There were no associated clinical or laboratory evidence of hemolysis.

  16. Strongly enhanced temperature dependence of the chemical potential in FeSe

    Science.gov (United States)

    Rhodes, L. C.; Watson, M. D.; Haghighirad, A. A.; Eschrig, M.; Kim, T. K.

    2017-05-01

    Employing a 10-orbital tight-binding model, we present a set of hopping parameters fitted directly to our latest high-resolution angle-resolved photoemission spectroscopy (ARPES) data for the high-temperature tetragonal phase of FeSe. Using these parameters, we predict a large 10 meV shift of the chemical potential as a function of temperature. To confirm this large temperature dependence, we performed ARPES experiments on FeSe and observed a ˜25 meV rigid shift to the chemical potential between 100 and 300 K. This strong shift has important implications for theoretical models of superconductivity and of nematic order in FeSe materials.

  17. Tumor Antigen-Dependent and Tumor Antigen-Independent Activation of Antitumor Activity in T Cells by a Bispecific Antibody-Modified Tumor Vaccine

    Directory of Open Access Journals (Sweden)

    Philippe Fournier

    2010-01-01

    Full Text Available New approaches of therapeutic cancer vaccination are needed to improve the antitumor activity of T cells from cancer patients. We studied over the last years the activation of human T cells for tumor attack. To this end, we combined the personalized therapeutic tumor vaccine ATV-NDV—which is obtained by isolation, short in vitro culture, irradiation, and infection of patient's tumor cells by Newcastle Disease Virus (NDV—with bispecific antibodies (bsAbs binding to this vaccine and introducing anti-CD3 (signal 1 and anti-CD28 (signal 2 antibody activities. This vaccine called ATV-NDV/bsAb showed the unique ability to reactivate a preexisting potentially anergized antitumor memory T cell repertoire. But it also activated naive T cells to have antitumor properties in vitro and in vivo. This innovative concept of direct activation of cancer patients' T cells via cognate and noncognate interactions provides potential for inducing strong antitumor activities aiming at overriding T cell anergy and tumor immune escape mechanisms.

  18. Antibody biotechnology

    African Journals Online (AJOL)

    STORAGESEVER

    2009-07-06

    Jul 6, 2009 ... and automated, the hybrid cells can be stored for many years in liquid nitrogen and antibodies production is homogeneous. The hybridoma method .... they may be modified to vehicle active molecules such as radio-isotopes, toxins, cytokines, enzyme etc. In these cases, the therapeutic effect is due to ...

  19. Catalytic Antibodies

    Indian Academy of Sciences (India)

    The ability of the highly evolved machinery of immune system to produce structurally and functionally complex ... to Pauling, if the structure of the antigen binding site of antibodies were to be produced in a random ..... where the immune system of the body is destructive, as in autoimmune disorders or after organ transplant.

  20. Catalytic Antibodies

    Indian Academy of Sciences (India)

    While chemistry provides the framework for understanding the structure and function of biomolecules, the immune sys- tem provides a highly evolved natural process to generate one class of complex biomolecules – the antibodies. A combination of the two could be exploited to generate new classes of molecules with novel ...

  1. Chloride ion-dependent surface-enhanced Raman scattering study of biotin on the silver surface

    International Nuclear Information System (INIS)

    Liu Fangfang; Gu Huaimin; Yuan Xiaojuan; Dong Xiao; Lin Yue

    2011-01-01

    In the present paper, the surface enhanced Raman scattering (SERS) technique was employed to study the SERS spectra of biotin molecules formed on the silver surface. The adsorption geometries of biotin molecules on the silver surface were analyzed based on the SERS data. It can be found that most vibration modes show a Raman shift in silver sol after the addition of sodium chloride solution. In addition, The Raman signals of biotin become weaker and weaker with the increase of the concentration of sodium chloride. This may be due to that the interaction between chloride ions and silver particles is stronger than the interaction between biotin molecules and silver particles. When the concentration of sodium chloride in silver colloid is higher than 0.05mol/L, superfluous chloride ions may form an absorption layer so that biotin can not be adsorbed on silver surface directly. The changes in intensity and profile shape in the SERS spectra suggest different adsorption behavior and surface-coverage of biotin on silver surface. The SERS spectra of biotin suggest that the contribution of the charge transfer mechanism to SERS may be dominant.

  2. Plasma-enhanced antibody immobilization for the development of a capillary-based carcinoembryonic antigen immunosensor using laser-induced fluorescence spectroscopy.

    Science.gov (United States)

    Yu, Qiaoling; Zhan, Xuefang; Liu, Kunping; Lv, Hao; Duan, Yixiang

    2013-05-07

    In this study, antibody immobilization using a microwave-induced H2O/Ar plasma pretreatment was achieved for the first time. Plasma was used to activate the surface of a capillary-based immunosensor by increasing the density of silicon hydroxyls and dangling bonds to ensure better silanization. The capture antibodies were covalently immobilized after the silanized surface reacted with glutaraldehyde and antibodies. A Cy3-labeled detection antibody was used in combination with the antigen captured by the immunosensor to complete the sandwich-type immunoassay, and the signals were measured using a laser-induced fluorescence system. Microwave-induced H2O/Ar plasma pretreatment of the carcinoembryonic antigen (CEA) immunosensor improved the antibody immobilization, and there was an obvious improvement in the linear detection range, i.e., 1 order of magnitude compared with a commercial enzyme-linked immunosorbent assay (ELISA). This novel immobilization method dramatically improved the detection limit (0.5 pmol/L CEA) and sensitivity. Assay validation studies indicated that the correlation coefficient reached 0.9978, and the relative standard deviations were Ar plasma was demonstrated to be a sensitive tool for CEA diagnostics.

  3. Temporal dependence of in vivo USPIO-enhanced MRI signal changes in human carotid atheromatous plaques

    Energy Technology Data Exchange (ETDEWEB)

    Tang, T.Y.; Sadat, U. [Cambridge University Hospitals NHS Foundation Trust, University Department of Radiology, Cambridge (United Kingdom); Cambridge University Hospitals NHS Foundation Trust, Cambridge Vascular Unit, Cambridge (United Kingdom); Patterson, A.J.; Graves, M.J.; Howarth, S.P.S.; U-King-Im, J.M.; Li, Z.Y.; Young, V.E.; Gillard, J.H. [Cambridge University Hospitals NHS Foundation Trust, University Department of Radiology, Cambridge (United Kingdom); Miller, S.R. [GlaxoSmithKline, Biostatistics and Data Sciences, Harlow (United Kingdom); Walsh, S.R.; Boyle, J.R.; Gaunt, M.E. [Cambridge University Hospitals NHS Foundation Trust, Cambridge Vascular Unit, Cambridge (United Kingdom)

    2009-07-15

    Ultrasmall superparamagnetic iron oxide (USPIO)-enhanced MRI has been shown to be a useful modality to image activated macrophages in vivo, which are principally responsible for plaque inflammation. This study determined the optimum imaging time-window to detect maximal signal change post-USPIO infusion using T{sub 1}-weighted (T{sub 1}w), T{sub 2}*-weighted (T{sub 2}*w) and quantitative T{sub 2}* (qT{sub 2}*) imaging. Six patients with an asymptomatic carotid stenosis underwent high resolution T{sub 1}w, T{sub 2}*w and qT{sub 2}* MR imaging of their carotid arteries at 1.5 T. Imaging was performed before and at 24, 36, 48, 72 and 96 h after USPIO (Sinerem trademark, Guerbet, France) infusion. Each slice showing atherosclerotic plaque was manually segmented into quadrants and signal changes in each quadrant were fitted to an exponential power function to model the optimum time for post-infusion imaging. The power function determining the mean time to convergence for all patients was 46, 41 and 39 h for the T{sub 1}w, T{sub 2}*w and qT{sub 2}* sequences, respectively. When modelling each patient individually, 90% of the maximum signal intensity change was observed at 36 h for three, four and six patients on T{sub 1}w, T{sub 2}*w and qT{sub 2}*, respectively. The rates of signal change decrease after this period but signal change was still evident up to 96 h. This study showed that a suitable imaging window for T{sub 1}w, T{sub 2}*w and qT{sub 2}* signal changes post-USPIO infusion was between 36 and 48 h. Logistically, this would be convenient in bringing patients back for one post-contrast MRI, but validation is required in a larger cohort of patients. (orig.)

  4. The dependence of Pi2 waveforms on periodic velocity enhancements within bursty bulk flows

    Directory of Open Access Journals (Sweden)

    K. R. Murphy

    2011-03-01

    Full Text Available Pi2s are a category of Ultra Low Frequency (ULF waves associated with the onset of magnetic substorms. Recent work has suggested that the deceleration of bulk plasma flows in the central plasmasheet, known as bursty bulk flows (BBFs, are able to directly-drive Pi2 oscillations. Some of these studies have further shown evidence that there is a one-to-one correlation between Pi2 magnetic waveforms observed on the ground and periodic peaks in flow velocity within the BBF, known as flow bursts. Utilising a favourable conjunction between the Geotail spacecraft and the Canadian Array for Real-time Investigations of Magnetic Activity (CARISMA magnetometer array on 31 May 1998, we examine the causality of the link between BBF flow bursts and Pi2 waveforms. Using a series of analytical tests in both the time and frequency domains, we find that while the Pi2 and BBF waveforms are very similar, the ground response for this event occurs prior to the observed flow enhancements in the magnetotail. We conclude that during this specific case study the temporal variations of the flow bursts within the BBF are not directly-driving the observed ground-based Pi2 waveforms, despite the fact that a visual inspection of both time-series might initially suggest that there is a causal relationship. We postulate that rather than there being a direct causal relation, the similar waveforms observed in both Pi2s and BBFs may result from temporal variations in a common source for both the BBFs and the Pi2s, such as magnetic reconnection in the tail, this source modulating both the Pi2 and BBF at the same frequency.

  5. Baseline-dependent effect of noise-enhanced insoles on gait variability in healthy elderly walkers.

    Science.gov (United States)

    Stephen, Damian G; Wilcox, Bethany J; Niemi, James B; Franz, Jason R; Franz, Jason; Kerrigan, Dr; Kerrigan, D Casey; D'Andrea, Susan E

    2012-07-01

    The purpose of this study was to determine whether providing subsensory stochastic-resonance mechanical vibration to the foot soles of elderly walkers could decrease gait variability. In a randomized double-blind controlled trial, 29 subjects engaged in treadmill walking while wearing sandals customized with three actuators capable of producing stochastic-resonance mechanical vibration embedded in each sole. For each subject, we determined a subsensory level of vibration stimulation. After a 5-min acclimation period of walking with the footwear, subjects were asked to walk on the treadmill for six trials, each 30s long. Trials were pair-wise random: in three trials, actuators provided subsensory vibration; in the other trials, they did not. Subjects wore reflective markers to track body motion. Stochastic-resonance mechanical stimulation exhibited baseline-dependent effects on spatial stride-to-stride variability in gait, slightly increasing variability in subjects with least baseline variability and providing greater reductions in variability for subjects with greater baseline variability (pgait variability for subjects with more variable gait. Stochastic-resonance mechanical vibrations may provide an effective intervention for preventing falls in healthy elderly walkers. Published by Elsevier B.V.

  6. Centrality dependence of strangeness enhancement in ultrarelativistic heavy ion collisions: A core-corona effect

    International Nuclear Information System (INIS)

    Aichelin, J.; Werner, K.

    2009-01-01

    In ultrarelativistic heavy ion collisions, the multiplicity of multistrange baryons per participating nucleon increases with centrality in a different fashion for different systems and energies. At RHIC, for copper+copper (CuCu) collisions the increase is much steeper than for gold-gold (AuAu) collisions. We show that this system size dependence is due to a core-corona effect: the relative importance of the corona as compared to the core (thermalized matter) contribution varies and the contribution of a corona nucleon to the multiplicity differs from that of a core nucleon. φ mesons follow--as all hadrons--the same trend, but the difference between core and corona multiplicity is relatively small, and therefore the CuCu and AuAu results are quite similar. This simple geometrical explanation makes also a strong case in favor of the validity of Glauber geometry in the peripheral regions of ultrarelativistic heavy ion collisions, which is crucial for understanding the early evolution of the system.

  7. Potent dried drug nanosuspensions for oral bioavailability enhancement of poorly soluble drugs with pH-dependent solubility.

    Science.gov (United States)

    Mou, Dongsheng; Chen, Huabing; Wan, Jiangling; Xu, Huibi; Yang, Xiangliang

    2011-07-15

    The objective of this study was to enhance the oral bioavailability of itraconazole (ITZ) with dried drug nanosuspensions. The feasibility of using poloxamer 407 or HPMC (50 cp) as stabilizers for preparing ITZ nanosuspensions by facile acid-base neutralization was investigated. Dried ITZ nanosuspensions were prepared by spray drying. The effect of matrix former on the dissolution rate of dried ITZ nanosuspensions was investigated. Results from dissolution test revealed that spray-dried ITZ nanosuspensions (ITZ:HPMC:mannitol 1:0.5:2, w/w) preserved the high dissolution rate from nanosuspensions. After oral administration in rats, the AUC(0-36) from dried ITZ nanosuspensions was 1.5-fold and 1.8-fold higher than the AUC(0-36) from sporanox pellets (commercial product) in the fed and fasted states, respectively (pnanosuspensions showed no difference between fed/fasted states, because this formulation could enhance the adsorption of ITZ in target site (small intestine) regardless of food intake. In addition, dried ITZ nanosuspensions showed a lower inter-individual variability in terms of bioavailability. Positive results demonstrate that dried drug nanosuspensions formulation prepared by acid-base neutralization combined with spray drying may be a promising method for enhancing the oral bioavailability of poorly soluble drugs with pH-dependent solubility. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Calculated shape dependence of electromagnetic field in tip-enhanced Raman scattering by using a monopole antenna model

    Science.gov (United States)

    Kitahama, Yasutaka; Itoh, Tamitake; Suzuki, Toshiaki

    2018-05-01

    To evaluate the shape of an Ag tip with regard to tip-enhanced Raman scattering (TERS) signal, the enhanced electromagnetic (EM) field and scattering spectrum, arising from surface plasmon resonance at the apex of the tip, were calculated using a finite-difference time domain (FDTD) method. In the calculated forward scattering spectra from the smooth Ag tip, the band appeared within the visible region, similar to the experimental results and calculation for a corrugated Ag cone. In the FDTD calculation of TERS, the Ag tip acting as a monopole antenna was adopted by insertion of a perfect electric conductor between the root of the tip and a top boundary surface of the calculation space. As a result, the EM field was only enhanced at the apex. The shape dependence i.e. the EM field calculated at the apex with various curvatures on the different tapered tips, obtained using the monopole antenna model, was different from that simulated using a conventional dipole antenna model.

  9. Social stress-enhanced severity of Citrobacter rodentium induced colitis is CCL2-dependent and attenuated by pro